Long-lived polynucleotide molecules

ABSTRACT

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of long-lived polynucleotides, primary transcripts and mmRNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/774,719, filed Sep. 11, 2015, which is a national stage entry ofPCT/US14/25225, filed Mar. 13, 2014, which claims priority to U.S.Provisional Patent Application No. 61/778,644, filed Mar. 13, 2013,entitled Long-Lived Polynucleotide Molecules and U.S. Provisional PatentApplication No. 61/828,784, filed May 30, 2013, entitled Long-LivedPolynucleotide Molecules, the contents of each of which are hereinincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web, and is herebyincorporated by reference in its entirety. Said ASCII copy, created onMar. 21, 2022, is named MRNA_027_03US_ST25.txt and is 66,822 bytes insize.

FIELD OF THE INVENTION

The invention relates to compositions, methods, processes, kits anddevices for the design, preparation, manufacture and/or formulation ofpolynucleotides encoding at least one longevity enhancing sequence suchas, but not limited to, carboxy-terminal peptide (CTP), albumin and/orIgG4 which may increase the length of time a polynucleotide, primaryconstruct and/or mmRNA remains in a cell, tissue and/or organism. In oneaspect, the invention relates to modified RNA in therapeutics. Themodified RNA of the invention may encode peptides, polypeptides ormultiple proteins. The modified RNA of the invention may also be used toproduce polypeptides of interest which may include at least onelongevity enhancing sequence such as, but not limited to,carboxy-terminal peptide (CTP), albumin and/or IgG4. The modified RNAmolecules of the invention may therefore be referred to as modifiedmRNA. The polypeptides of interest may be used in therapeutics and/orclinical and research settings.

BACKGROUND OF THE INVENTION

Polynucleotides are susceptible to denaturation or enzymatic degradationin cells, tissues and/or organisms (e.g., in the blood, liver orkidney). Accordingly, polynucleotides have short circulatory half-livessuch as, but not limited to, several hours. Because of their lowstability, the polynucleotides may need to be administered repeatedly orcontinuously as to maintain a desired concentration of the encodedpolypeptide in the cell, tissue and/or organism.

Thus, there is a need for technologies that will alter (e.g., prolongand/or shorten) the half-lives of polynucleotides while maintaining apharmacological efficacy thereof. It would also be desirable to havepolynucleotides which may be therapeutics to have enhanced serumstability, high activity and a low probability of inducing an undesiredimmune response when administered or delivered to a cell, tissue and/ororganism.

Unfavorable pharmacokinetics of therapeutics, such as a short serumhalf-life, can delay the development of many otherwise promisingpolynucleotide therapeutics. Serum half-life is an empiricalcharacteristic of a molecule, and must be determined experimentally foreach new potential therapeutic. For example, physiological clearancemechanism (e.g., renal filtration) can make the maintenance of desiredlevels of a polynucleotide therapeutic undesirable and/or unfeasiblebecause of cost or frequency of the required dosing regimen. Conversely,a long serum half-life may be undesirable in some instances where atherapeutic or its metabolites has toxic side effects.

The present invention addresses both the problem of denaturation and/ordegradation of polynucleotides by providing nucleic acid based compoundsor polynucleotides which encode a polypeptide of interest (e.g.,modified mRNA or mmRNA) and a longevity enhancing sequence such as, butnot limited to, carboxy-terminal peptide (CTP), albumin and IgG4 andwhich may have structural and/or chemical features that avoid one ormore of the problems in the art.

To this end, the inventors have shown that certain modified mRNAsequences have the potential as therapeutics with benefits beyond justevading, avoiding or diminishing the immune response. Such studies aredetailed in published co-pending applications International PublicationNo WO2012019168 filed Aug. 5, 2011, International Publication NoWO2012045082 filed Oct. 3, 2011, and International Publication NoWO2012045075 filed Oct. 3, 2011, the contents of which are incorporatedherein by reference in their entirety.

SUMMARY OF THE INVENTION

Described herein are compositions, methods, processes, kits and devicesfor the design, preparation, manufacture and/or formulation ofpolynucleotides encoding at least one longevity enhancing sequence suchas, but not limited to, carboxy-terminal peptide (CTP), albumin andIgG4. Such polynucleotides may be chemically modified mRNA (mmRNA)molecules.

In one aspect, provided herein is an isolated mRNA polynucleotide. Theisolated mRNA polynucleotide may encode a polypeptide of interest and afirst longevity enhancing sequence such as, but not limited to, acarboxy-terminal peptide, an albumin sequence and an IgG4 sequence. Theisolated mRNA polynucleotide may be codon optimized. Also providedherein is a composition comprising at least one isolated mRNApolynucleotide.

The carboxy-terminal peptide may be located at a position such as, butnot limited to, a region that is located between the 5′ terminus tocoding region of a polypeptide of interest such as, but not limited to,the 5′UTR, within coding region and a region that is located between thecoding region of the polypeptide of interest to 3′ terminus such as, butnot limited to, the 3′UTR.

The poly-A tail of the isolated mRNA polynucleotide may be approximately140 to 160 nucleotides in length. The 5′cap structure may be selectedfrom, but is not limited to, Cap0, Cap1, ARCA, inosine,N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine. The at least one isolated polynucleotide may also bepurified.

The isolated mRNA polynucleotide may also encode a second, third or morecarboxy-terminal peptides. The at least one isolated mRNA polynucleotidemay encode the same amino acid sequence for the first and second, thirdor more carboxy-terminal peptides. The at least one isolated mRNApolynucleotide may encode the same nucleic acid sequence for the firstand second, third or more carboxy-terminal peptides. In one aspect, theat least one isolated mRNA polynucleotide encodes a nucleic acidsequence for the first carboxy-terminal peptide and the secondcarboxy-terminal peptide has a nucleic acid sequence at least 85%homologous with the nucleic acid sequence for the first carboxy-terminalpeptide. The carboxy-terminal peptides of the present invention mayinclude, but are not limited to, those recited in SEQ ID NOs: 1-3 andportion, fragments and variants thereof.

In one aspect the first carboxy-terminal peptide, the secondcarboxy-terminal peptide and the third carboxy-terminal peptide may beeach located at a position selected from a region that is locatedbetween the 5′ terminus to coding region of a polypeptide of interestsuch as, but not limited to, the 5′UTR, the coding region and a regionthat is located between the coding region of the polypeptide of interestto 3′ terminus such as, but not limited to, the 3′UTR. As a non-limitingexample, the first carboxy-terminal peptide is located at the positionof 5′ terminus to coding region of a polypeptide of interest. Further,the second carboxy-terminal peptide and the third carboxy-terminalpeptide may be located at the position coding region of the polypeptideof interest to 3′ terminus. As another non-limiting example, the firstcarboxy-terminal peptide is located in the 5′UTR. Further, the secondcarboxy-terminal peptide and the third carboxy-terminal peptide may belocated in the 3′UTR.

The composition may comprise at least a first modified nucleoside. Inone aspect the at least one isolated mRNA polynucleotide comprises atleast two modifications. The at least two modification may be located onone or more of a nucleoside and/or a backbone linkage betweennucleosides. The one or more backbone linkages may be modified byreplacement of one or more oxygen atom and/or replacing at least onebackbone linkage with a phosphorothioate linkage. The one or moremodifications may be located on a sugar of one or more nucleosides.

In one aspect, the one or more modified nucleosides may include, but arenot limited to, pyridin-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

In one aspect, the at least one modification is located on one or morenucleobases. The one or more nucleobases are selected from the groupconsisting of cytosine, guanine, adenine, thymine and uracil.

In one aspect of the present invention, provided is a method ofincreasing the half-life of an mRNA polynucleotide in a cell, tissueand/or organism comprising contacting the cell, tissue and/or organismwith the composition or isolated mRNA described herein.

In one aspect of the present invention, provided is a method ofincreasing the half-life of protein in a cell, tissue and/or organismcomprising contacting the cell, tissue and/or organism with the isolatedmRNA described herein. The isolated mRNA may be codon optimized and maycomprise a chemical modification.

In one aspect of the present invention, provided is a method ofincreasing the level of protein in a cell, tissue and/or organismcomprising contacting the cell, tissue and/or organism with thecomposition or isolated mRNA described herein. The isolated mRNA may becodon optimized and may comprise a chemical modification.

In one aspect of the present invention, provided is a pharmaceuticalcomposition comprising the isolated mRNA described herein and apharmaceutically acceptable excipient. The isolated mRNA may be codonoptimized and may comprise a chemical modification.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of a primary construct of the present invention.

FIG. 2 is a schematic of a bicistronic primary construct of the presentinvention.

FIG. 3 is a schematic of a primary construct of the present inventionwith a longevity enhancing sequence.

FIG. 4 is a schematic of a primary construct of the present inventionwith a longevity enhancing sequence.

FIG. 5 is a schematic of a primary construct of the present inventionwith a longevity enhancing sequence.

FIG. 6 is a schematic of a primary construct of the present inventionwith a longevity enhancing sequence.

FIG. 7 is clone map useful in the present invention.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo,in situ or ex vivo, such as to cause intracellular translation of thenucleic acid and production of an encoded polypeptide of interest. Ofparticular importance is the delivery and function of a non-integrativepolynucleotide.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides encoding one or more polypeptidesof interest and further encoding at least one carboxy-terminal peptide(CTP). Also provided are systems, processes, devices and kits for theselection, design and/or utilization of the polynucleotides encoding thepolypeptides of interest further encoding at least one carboxy-terminalpeptide (CTP) described herein.

According to the present invention, these polynucleotides are preferablymodified as to avoid the deficiencies of other polypeptide-encodingmolecules of the art. Hence these polynucleotides are referred to asmodified mRNA or mmRNA.

Provided herein, in part, are polynucleotides, primary constructs and/ormmRNA encoding polypeptides of interest which have been designed toimprove one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, mRNA half-life, translationefficiency, immune evasion, protein production capacity, secretionefficiency (when applicable), accessibility to circulation, proteinhalf-life and/or modulation of a cell's status, function and/oractivity. Specifically, the polynucleotides, primary constructs and/ormmRNA of the present invention are useful in altering the longevity ofpolynucleotides in a cell, tissue and/or organism.

I. Compositions of the Invention

The present invention provides nucleic acid molecules, specificallypolynucleotides, primary constructs and/or mmRNA which encode one ormore polypeptides of interest and further encode at least one longevityenhancing sequence such as, but not limited to, a carboxy-terminalpeptide, albumin and IgG4. As used herein, “carboxy-terminal peptides”or “CTPs” are peptide moieties which, when fused to or incorporated intoother peptides or proteins result in an increase in half-life of theresultant protein. These long-acting peptides can, consequently resultin therapeutic benefit to the organism treated with such resultantpeptides or proteins or alternatively when treated with one or morepolynucleotides, primary constructs or mmRNA which encode such proteinshaving CTPs. The polynucleotides, primary constructs and/or mmRNA mayencode at least one carboxy-terminal peptide. As a non-limiting example,the polynucleotides, primary constructs and/or mmRNA may encode acarboxy-terminal peptide attached at the amino terminus of the encodedpolypeptide of interest. In another non-limiting example, thepolynucleotides, primary constructs and/or mmRNA may encode acarboxy-terminal peptide attached to the amino and carboxy terminus ofthe encoded polypeptide of interest.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAencoded a polypeptide of interest and encode at least one full-lengthcarboxy-terminal peptide. In another embodiment, the polynucleotides,primary constructs and/or mmRNA encoded a polypeptide of interest andencode at least one truncated carboxy-terminal peptide. In yet anotherembodiment, the polynucleotides, primary constructs and/or mmRNA encodeda polypeptide of interest and encode at least one full-lengthcarboxy-terminal peptide and at least one truncated carboxy-terminalpeptide.

In one embodiment, the half-life of a polynucleotide in a cell, tissueand/or organism is increased by providing the cell, tissue and/ororganism with a polynucleotide, primary construct and/or mmRNAcomprising at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide (CTP). In one embodiment, the at least onecarboxy-terminal peptide (CTP) may be attached to the carboxy terminusof a polypeptide of interest. In another embodiment, the at least onecarboxy-terminal peptide (CTP) may be attached to the amino terminus ofthe polypeptide of interest.

In one embodiment, the polynucleotide, primary construct and/or mmRNAmay comprise at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide (CTP) located on the 5′ end of the flankingregion, after the 5′ terminal cap, in the 5′UTR, before the firstoperational region, after the first operational region, within theflanking region, after the first operational region, prior to the signalsequence region, after the signal sequence region, before the firstregion of linked nucleosides, after the first region of linkednucleosides, within the first region of linked nucleosides, before thesecond operational region, after the second operational region, beforethe stop codon, after the stop codon, before the second operationalregion, after the second operational region, before the second flankingregion, after the second flanking region, within the second flankingregion, within the 3′UTR, before the 3′ tailing sequence, after the 3′tailing sequence, within the 3′ tailing sequence and combinationthereof.

In one embodiment, the polynucleotide, primary construct and/or mmRNAmay comprise at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide at the 5′ end of the polynucleotide, primaryconstruct and/or mmRNA.

In one embodiment, the carboxy-terminal peptide may be located after thesignal peptide sequence.

In one embodiment, the polynucleotide, primary construct and/or mmRNAmay comprise at least two nucleic acid sequences encoding at least onecarboxy-terminal peptide at the 3′ end of the polynucleotide, primaryconstruct and/or mmRNA. The carboxy-terminal peptides located at the 3′end of the polynucleotide, primary construct and/or mmRNA may be thesame or different. In another aspect, the nucleic acid sequence encodingthe carboxy-terminal peptides located at the 3′ end of thepolynucleotide, primary construct and/or mmRNA may be different but theyencode the same carboxy-terminal peptide.

In one embodiment, the polynucleotide, primary construct and/or mmRNAcomprises at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide at the 5′ end of the polynucleotide, primaryconstruct and/or mmRNA and at least two nucleic acid sequences encodingat least one carboxy-terminal peptide at the 3′ end of thepolynucleotide, primary construct and/or mmRNA. In one aspect, thecarboxy-terminal peptide at the 5′ end may be the same as at least oneof the at least two carboxy-terminal peptides located at the 3′ end. Inanother aspect, the carboxy-terminal peptide at the 5′ end may be afragment of at least one of the carboxy-terminal peptides located at the3′ end. In one aspect, the nucleic acid sequence encoding thecarboxy-terminal peptides located at the 5′ end of the polynucleotide,primary construct and/or mmRNA may be the same or different from thenucleic acid sequence encoding the carboxy-terminal peptide located atthe 3′ end. As a non-limiting example, the carboxy-terminal peptide atthe 5′end is located after the signal peptide before the sequenceencoding the protein of interest and the carboxy-terminal peptides atthe 3′end is located after the region encoding the protein of interest.

In one embodiment, the polynucleotide, primary construct and/or mmRNAcomprising at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide (CTP) may include at least one chemicalmodification described herein and/or known in the art.

In one embodiment, the polynucleotide, primary construct and/or mmRNAmay comprise at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide such as, but not limited to, chorionicgonadotrophin carboxy-terminal peptide or beta-gonadotrophin. In oneembodiment, the encoded carboxy-terminal peptide is a variant of thenative carboxy-terminal peptide. In one embodiment, the encodedcarboxy-terminal peptide may differ from the native carboxy-terminalpeptide by 1, 2, 3, 4, 5 or more conservative amino acid substitutions.As a non-limiting example, the encoding carboxy-terminal peptide may bea variant of chorionic gonadotrophin carboxy-terminal peptide by one tofive conservative amino acid substitutions as described in U.S. Pat. No.5,712,122, the contents of which are herein incorporated by reference inits entirety. As another non-limiting example, Table 1 is a listing ofcarboxy-terminal peptides which may be used in the present invention.

TABLE 1 Carboxy-Terminal Peptides SEQ ID Identifier Sequence NO CTP-001SSSSKAPPPSLPSPSRLPGPSDTPILPQ 1 CTP-002 SSSSKAPPPSLP 2 CTP-003DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILQ 3

In one embodiment, the encoded carboxy-terminal peptide may differ fromthe native carboxy-terminal peptide by 1, 2, 3, 4, 5 or more radicalamino acid substitutions.

In one embodiment, the encoded carboxy-terminal peptide may have 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to the nativecarboxy-terminal peptide.

In one embodiment, a cell, tissue and/or organism may be contacted withthe polynucleotide, primary construct and/or mmRNA comprising at leastone nucleic acid sequence encoding at least one carboxy-terminal peptidein order to produce a polypeptide of interest the cell, tissue and/ororganism. In another embodiment, a cell, tissue and/or organism may becontacted with the polynucleotide, primary construct or mmRNA in orderto increase the level of a polypeptide of interest in the cell, tissueand/or organism.

In one embodiment, the carboxy-terminal peptide may be glycosylated.

In another embodiment, the carboxy-terminal peptide may be truncated.

In one embodiment, a polynucleotide, primary construct and/or mmRNA maycomprise carboxy-terminal peptide derived from chorionic gonadotrophin.As a non-limiting example, the polynucleotide, primary construct and/ormmRNA may encode a polypeptide comprising at least one carboxy-terminalpeptide of chorionic gonadotrophin as described in US Patent PublicationNo. US20130243747 or U.S. Pat. No. 8,476,234, the contents of each ofwhich are herein incorporated by reference in their entirety. As anothernon-limiting example, the polynucleotide, primary construct and/or mmRNAmay encode a carboxy-terminal peptide having an amino acid sequence suchas SEQ ID NO: 1 and SEQ ID NO: 2 described in US Patent Publication No.US20130243747, the contents of which are herein incorporated byreference in its entirety. As another non-limiting example, thepolynucleotide, primary construct and/or mmRNA may encode acarboxy-terminal peptide having an amino acid sequence such as SEQ IDNO: 1 and SEQ ID NO: 2 described in U.S. Pat. No. 8,476,234, thecontents of which are herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay encode a polypeptide comprising at least one carboxy-terminalpeptide of chorionic gonadotrophin as described in US Patent PublicationNo. US20130243747, the contents of which are herein incorporated byreference in its entirety, where the carboxy-terminal peptide isattached to the carboxy terminus but not to the amino terminus of apolypeptide. As another non-limiting example, the polynucleotides,primary constructs and/or mmRNA encoding a polypeptide comprising atleast one carboxy-terminal peptide of chorionic gonadotrophin may bemade by the methods described in US Patent Publication No. US20130243747and U.S. Pat. No. 8,476,234, the contents of each of which are hereinincorporated by reference in their entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay encode a polypeptide comprising at least two carboxy-terminalpeptides (CTP) of chorionic gonadotrophin. As a non-limiting example,the carboxy-terminal peptides of chorionic gonadotrophin may be thosedescribed in US Patent Publication No. US20130184207, the contents ofwhich are herein incorporated by reference in its entirety. As anothernon-limiting example, the carboxy-terminal peptides of chorionicgonadotrophin may comprise the amino acid sequence SEQ ID NO: 17 or theamino acid sequence SEQ ID NO: 18 described in US Patent Publication No.US20130184207, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, a polynucleotide, primary construct and/or mmRNA mayencode a binding domain which is capable of binding to serum albuminwhich may increase the half-life of the polypeptide. As a non-limitingexample, the polynucleotides, primary constructs and/or mmRNA may encodea polypeptide comprising a binding domain which is capable of binding toserum albumin as described in PCT Application No. WO2013128027, thecontents of which are herein incorporated by reference in its entirety.In another non-limiting example, the polynucleotides, primary constructsand/or mmRNA may encode a polypeptide comprising a binding domain whichis capable of binding to serum albumin. The binding domain capable ofbinding to serum albumin may be attached to the carboxy terminus but notto the amino terminus of a polypeptide, such as the binding domaindescribed in PCT Application No. WO2013128027, the contents of which areherein incorporated by reference in its entirety. As anothernon-limiting example, the binding domain of serum albumin is derivedfrom a combinatorial library or an antibody binding domain, as describedin PCT Application WO2013128027, the contents of which are hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mRNAmay encode a polypeptide comprising a binding domain which is capable ofbinding to serum albumin, wherein the binding domain comprises between10 and 25 amino acid residues as described in PCT Application No.WO2013128027, the contents of which are herein incorporated by referencein its entirety. In another embodiment, the polynucleotides, primaryconstructs and/or mmRNA may encode a polypeptide comprising a bindingdomain which is capable of binding to serum albumin wherein the bindingdomain capable of binding to serum albumin comprises the amino acidsequence Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 50),wherein Xaa is any amino acid, as described in PCT Application No.WO2013128027, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay encode a polypeptide comprising a binding domain which is capable ofbinding to serum albumin, wherein the binding domain capable of bindingto serum albumin is derived from a CDR of a single domain antibody, asdescribed in PCT Application No. WO2013128027, the contents of which areherein incorporated by reference in its entirety. As a non limitingexample, the polynucleotides, primary constructs and/or mmRNA may encodea polypeptide comprising a binding domain which is capable of binding toserum albumin, wherein the binding domain is binding to serum albuminwith an affinity (KD) of <500 nM, as described in PCT Application No.WO2013128027, the contents of which are herein incorporated by referencein its entirety. As another non limiting example, the polynucleotides,primary constructs and/or mmRNA may encode a binding domain which iscapable of binding to serum albumin having an amino acid sequence suchas in SEQ ID NOs: 51, 52, 54, 55, 57, 58, 60, 61, 75, 76, 81, 82, 85,86, 90, 91, 85, 96, 100 or 101 described in PCT Application No.WO2013128027, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, a polynucleotide, primary construct and/or mmRNA maycomprise a carboxy-terminal peptide derived from albumin to stabilizethe polypeptide. In one embodiment, the polynucleotides, primaryconstructs and/or mmRNA may encode a polypeptide comprising albumin,albumin fusion proteins or a fragment or variant of albumin that issufficient to stabilize and/or prolong the activity of the polypeptide.As a non-limiting example, the polynucleotides, primary constructsand/or mmRNA may encode a polypeptide comprising albumin, albumin fusionproteins or a fragment or variant of albumin as described in US PatentPublication No. US20130266553, the contents of which are hereinincorporated by reference in its entirety. As another non-limitingexample, the polynucleotides, primary constructs and/or mmRNA may encodea polypeptide derived from serum albumin comprising an amino acidsequence which is at least 95% identical SEQ ID NO:18 or comprisingamino acids 1 to 585 of SEQ ID NO:18 or comprising and amino acids 1 to387 of SEQ ID NO: 18 as described in US Patent Publication No.US20130266553, the contents of which are herein incorporated byreference in its entirety. As another non-limiting example, thepolynucleotides, primary constructs and/or mmRNA may encode apolypeptide comprising serum albumin or a fragment or variant of albuminwhich has been made by the methods described in US Patent PublicationNo. US20130243747, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, polynucleotides, primary constructs and/or mmRNA mayencode a polypeptide comprising an Fc domain, a CTP domain, an Fc-CTPdomain, or a combination thereof to increase half-life of thepolypeptide. As a non-limiting example, the polynucleotides, primaryconstructs and/or mmRNA may encode a polypeptide comprising at least onecarboxy-terminal or N-terminal peptide domain (e.g., Fe, CTP or Fc-CTP)as described in PCT Patent Publication No. WO2013152351, the contents ofwhich are herein incorporated by reference in its entirety. As anon-limiting examples, the polynucleotides, primary constructs and/ormmRNA may encode a polypeptide comprising an Fe domain which is about95% identical to SEQ ID NO: 1, described in PCT Patent Publication No.WO2013152351, the contents of which are herein incorporated by referencein its entirety. As another non-limiting example, the polynucleotides,primary constructs and/or mmRNA may encode a polypeptide comprising aCTP domain which is about 95% identical to SEQ ID NO: 3, described inPCT Patent Publication No. WO2013152351, the contents of which areherein incorporated by reference in its entirety. As anothernon-limiting example, the polynucleotides, primary constructs and/ormmRNA may encode a polypeptide comprising a Fc-CTP domain which is about95% identical to SEQ ID NO: 1, described in PCT Patent Publication No.WO2013152351, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay encode a polypeptide comprising a CTP domain, Fe domain, Fc-CTPdomain, or combination thereof fused to the C-terminus or the N terminusor both the N and the C terminus of a polypeptide such as thepolypeptides described in PCT Patent Publication No. WO2013152351, thecontents of which are herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAencoded a polypeptide of interest and encode at least one albuminsequence. In another embodiment, the polynucleotides, primary constructsand/or mmRNA encoded a polypeptide of interest and encode at least oneIgG4 sequence.

In one embodiment, the half-life of a polynucleotide in a cell, tissueand/or organism is increased by providing the cell, tissue and/ororganism with a polynucleotide, primary construct and/or mmRNAcomprising at least one nucleic acid sequence encoding at least onealbumin sequence and/or at least one IgG4 sequence. In one embodiment,the at least one albumin sequence and/or at least one IgG4 sequence maybe attached to the carboxy terminus of a polypeptide of interest. Inanother embodiment, the at least one albumin sequence and/or at leastone IgG4 sequence may be attached to the amino terminus of thepolypeptide of interest.

In one embodiment, the polynucleotide, primary construct and/or mmRNAmay comprise at least one nucleic acid sequence encoding at least onealbumin sequence and/or at least one IgG4 sequence located on the 5′ endof the flanking region, after the 5′ terminal cap, in the 5′UTR, beforethe first operational region, after the first operational region, withinthe flanking region, after the first operational region, prior to thesignal sequence region, after the signal sequence region, before thefirst region of linked nucleosides, after the first region of linkednucleosides, within the first region of linked nucleosides, before thesecond operational region, after the second operational region, beforethe stop codon, after the stop codon, before the second operationalregion, after the second operational region, before the second flankingregion, after the second flanking region, within the second flankingregion, within the 3′UTR, before the 3′ tailing sequence, after the 3′tailing sequence, within the 3′ tailing sequence and combinationthereof. As a non-limiting example, the albumin sequence is located inthe 3′UTR. As another non-limiting example, the IgG4 sequence is locatedin the 3′UTR.

In one embodiment, a cell, tissue and/or organism may be contacted withthe polynucleotide, primary construct and/or mmRNA comprising at leastone nucleic acid sequence encoding at least one carboxy-terminal peptidein order to maintain the level of a polypeptide of interest in the cell,tissue and/or organism.

In one embodiment, the present invention provides a method for reducingthe frequency of dosing of a cell, tissue and/or organism in order toproduce the polypeptide of interest. The method may comprise contactingthe cell, tissue and/or organism with a polynucleotide, primaryconstruct and/or mmRNA comprising at least one nucleic acid sequenceencoding at least one carboxy-terminal peptide.

In one embodiment, the present invention provides a method forprotecting polynucleotides, primary constructs and/or mmRNA againstdegradation. The method may comprise the addition of at least onenucleic acid sequence encoding at least one carboxy-terminal peptide inthe polynucleotide, primary construct and/or mmRNA.

In one embodiment, the present invention provides a method for extendingcirculatory half-life of polynucleotides, primary constructs and/ormmRNA. The method may comprise the addition of at least one nucleic acidsequence encoding at least one carboxy-terminal peptide in thepolynucleotide, primary construct and/or mmRNA.

In one embodiment, the present invention provides a method for enhancingthe potency of polynucleotides, primary constructs and/or mmRNA. Themethod may comprise the addition of at least one nucleic acid sequenceencoding at least one carboxy-terminal peptide in the polynucleotide,primary construct and/or mmRNA.

In one embodiment, the carboxy-terminal peptide encoded by the nucleicacid sequence is a full-length carboxy-terminal peptide. In anotherembodiment, the carboxy-terminal peptide encoded by the nucleic acidsequence is a truncated carboxy-terminal peptide.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAcomprise at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide before the coding region of the polypeptide ofinterest. In another embodiment, the polynucleotides, primary constructsand/or mmRNA comprise at least one nucleic acid sequence encoding atleast one carboxy-terminal peptide after the coding region of thepolypeptide of interest.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAcomprise at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide before the coding region of the polypeptide ofinterest and at least one nucleic acid sequence encoding at least onecarboxy-terminal peptide after the coding region of the polypeptide ofinterest. In another embodiment, the polynucleotide, primary constructsand/or mmRNA comprise one nucleic acid sequence encoding acarboxy-terminal peptide before the coding region of the polypeptide ofinterest and two nucleic acid sequences encoding at least onecarboxy-terminal peptide after the coding region of the polypeptide ofinterest.

In one embodiment, the nucleic acid sequences may encode the same and/ordifferent carboxy-terminal peptides. As a non-limiting example, thenucleic acid sequence encoding the carboxy-terminal peptide before thecoding region of the polypeptide of interest may encode the samecarboxy-terminal peptide as one of the nucleic acid sequences after thecoding region of the polypeptide of interest.

In another embodiment, the nucleic acid sequences may be different andencode the same carboxy-terminal peptide. As a non-limiting example, thenucleic acid sequence encoding the carboxy-terminal peptide before thecoding region of the polypeptide of interest may encode the samecarboxy-terminal peptide as one of the nucleic acid sequences after thecoding region of the polypeptide of interest, but the two nucleic acidsequences may have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100%sequence identity.

In one embodiment, the polynucleotides, primary constructs and mmRNA ofthe present invention have an altered property such as, but not limitedto, increased circulating half-life, increased plasma residence time,decreased clearance and increased clinical activity in a cell, tissueand/or organism.

In one embodiment, the polynucleotides, primary constructs and mmRNA ofthe present invention have an altered property such as, but not limitedto, improved potency, improved stability, elevated AUC levels andenhanced circulating half-life.

In one embodiment, the polynucleotides, primary constructs and mmRNA maycomprise at least one nucleic acid sequence encoding a carboxy-terminalpeptide which causes the polynucleotides, primary constructs and mmRNAto be poorly vascularized. As a non-limiting example, when acarboxy-terminal peptide is added to a follicle stimulating hormone(FSH) the resulting ovarian follicles appear to be poorly vascularized.If this effect is undesired a polynucleotide, primary construct or mmRNAencoding a helper molecule may be administered with the poorlyvascularized polynucleotides, primary constructs and mmRNA comprising atleast one nucleic acid sequence encoding a carboxy-terminal peptide. Thehelper molecule may be, but is not limited to, vascular endothelialgrowth factor (VEGF).

In one aspect, the helper molecule may be administered concurrently withthe poorly vascularized polynucleotide, primary construct or mmRNA. Theconcurrent administration may be from, but is not limited to, contactinga cell, tissue or organism with a formulation comprising both the helpmolecule and the poorly vascularized polynucleotide, primary constructor mmRNA or from contacting a cell, tissue or organism with two separateformulations of the helper molecule and the poorly vascularizedpolynucleotides, primary constructs or mmRNA.

In another aspect, the helper molecule and the poorly vascularizedpolynucleotide, primary construct or mmRNA may be administered to acell, tissue or organism on the same polynucleotide, primary constructor mmRNA by using a bi-cistronic or multi-cistronic polynucleotide,primary construct or mmRNA. As a non-limiting example, the bi-cistronicpolynucleotide, primary construct or mmRNA may comprise a nucleic acidsequence encoding a follicle stimulating hormone and a carboxy-terminalpeptide and also a nucleic acid sequence encoding VEGF. The twosequences may be, for example, separated by an IRES sequence, a proteincleavage site and/or a 2A peptide.

Carboxy-terminal peptides or C-terminal peptides useful in the presentinvention include those disclosed in Japanese and/or Chinese patentsJP04699991B2, JP04763616B2, JP04871124B2, JP04897814B2, JP04933439B2,JP03946638B2, JP04741086B2, JP03868740B2, CN102656182A, JP04718459B2,CN102639144A, JP03045539B2, JP04081137B2, JP04156024B2, JP04255618B2,and JP04334615B2, each of which is herein incorporated by reference inits entirety.

Proteins and or polypeptides having CTP moieties, and the specific CTPmoieties which may be encoded by the polynucleotides, primary constructsor mmRNA of the present invention include those disclosed in, forexample, Canadian patent or patent applications: CA2053864C,CA2118320A1, CA2120358C, CA2132868C, CA2160800A1, CA2160800C,CA2173750A1, CA2173750C, CA2208420A1, CA2208420C, CA2231348A1,CA2302647A1, CA2308571A1, CA2310713A1, CA2321677A1, CA2347062A1,CA2361840A1, CA2372582A1, CA2394572C, CA2400908A1, CA2400908C,CA2457067A1, CA2457849A1, CA2457851A1, CA2485365A1, CA2517487A1,CA2518903A1, CA2526099A1, CA2544333A1, CA2579131A1, CA2585507A1,CA2612613A1, CA2639115A1, CA2641342A1, CA2673222A1, CA2685437A1,CA2700662A1, CA2707352A1, CA2713386A1, CA2725551A1, CA2730996A1,CA2755927A1, CA2767503A1 and CA2781132A1; German patent or patentapplications: DE10235248A1, DE60117919T2, DE69029799T2 and DE69328831T2;European patent or patent applications: EP1012290A1, EP1032688A1,EP1032688B1, EP1054018A1, EP1054018B1, EP1064382B1, EP1087985A1,EP1113814A1, EP1123315A1, EP1263948A2, EP1276765A2, EP1276765B1,EP1285665A1, EP1316561A1, EP1316561B1, EP1319712A2, EP1319712B1,EP1342730A1, EP1342730B1, EP1425032A2, EP1425033A2, EP1425033B1,EP1434600A2, EP1434600B1, EP1490386A2, EP1610822A1, EP1610822B1,EP1624893A2, EP1638595A1, EP1673105A1, EP1673105B1, EP1697412A1,EP1697412B1, EP1802645A2, EP1809663A1, EP1809663B1, EP1951395A1,EP1951395B1, EP1991578A2, EP2001903A2, EP2016951A1, EP2016951B1,EP204566B1, EP2050762A2, EP2067788A2, EP2099913A1, EP2099913B1,EP2134165A2, EP2134165B1, EP2164508A2, EP2219607A1, EP2219607B1,EP2244717A1, EP224574A1, EP2249869A1, EP2249869B1, EP2313431A2,EP2325194A1, EP2364162A2, EP2412385A1, EP2414380A2, EP2451473A2,EP2471807A1, EP2504350A1, EP2532674A1, EP2532675A1, EP329699A1,EP461200A1, EP461200B1, EP607297A1, EP607297B1, EP636171A1, EP636171B1,EP644774A1, EP692259A2, EP695307A1, EP695307B1, EP725795A1, EP725795B1,EP793675A1, EP853945A1, EP853945B1 and EP882234A1; 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CTPs may range from 2-200 amino acids and may therefore be encoded bypolynucleotides, primary constructs or mmRNA ranging from 6-600nucleotides.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprise a polymer of nucleotides. These polymersare often referred to as polynucleotides. Exemplary nucleic acids orpolynucleotides of the invention include, but are not limited to,ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

In preferred embodiments, the polynucleotide or nucleic acid molecule isa messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA)refers to any polynucleotide which encodes a polypeptide of interest andwhich is capable of being translated to produce the encoded polypeptideof interest in vitro, in vivo, in situ or ex vivo. Polynucleotides ofthe invention may be mRNA or any nucleic acid molecule and may or maynot be chemically modified.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingpolynucleotides or primary RNA constructs which maintain a modularorganization, but which comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide including, in some embodiments, the lack of a substantialinduction of the innate immune response of a cell into which thepolynucleotide is introduced. As such, modified mRNA molecules of thepresent invention are termed “mmRNA.” As used herein, a “structural”feature or modification is one in which two or more linked nucleotidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide polynucleotide, primary construct or mmRNA withoutsignificant chemical modification to the nucleotides themselves. Becausechemical bonds will necessarily be broken and reformed to effect astructural modification, structural modifications are of a chemicalnature and hence are chemical modifications. However, structuralmodifications will result in a different sequence of nucleotides. Forexample, the polynucleotide “ATCG” may be chemically modified to“AT-5meC-G”. The same polynucleotide may be structurally modified from“ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted,resulting in a structural modification to the polynucleotide.

Polynucleotide, Primary Construct or mmRNA Architecture

The polynucleotides of the present invention are distinguished from wildtype mRNA in their functional and/or structural design features whichserve to, as evidenced herein, overcome existing problems of effectivepolypeptide production using nucleic acid-based therapeutics.

FIG. 1 shows a representative primary construct 100 of the presentinvention. As used herein, the term “primary construct” or “primary mRNAconstruct” refers to a polynucleotide transcript which encodes one ormore polypeptides of interest and which retains sufficient structuraland/or chemical features to allow the polypeptide of interest encodedtherein to be translated. Primary constructs may be polynucleotides ofthe invention. When structurally or chemically modified, the primaryconstruct may be referred to as a mmRNA.

Returning to FIG. 1 , the primary construct 100 here contains a firstregion of linked nucleotides 102 that is flanked by a first flankingregion 104 and a second flaking region 106. As used herein, the “firstregion” may be referred to as a “coding region” or “region encoding” orsimply the “first region.” This first region may include, but is notlimited to, the encoded polypeptide of interest. The polypeptide ofinterest may comprise at its 5′ terminus one or more signal peptidesequences encoded by a signal peptide sequence region 103. The flankingregion 104 may comprise a region of linked nucleotides comprising one ormore complete or incomplete 5′ UTRs sequences. The flanking region 104may also comprise a 5′ terminal cap 108. The second flanking region 106may comprise a region of linked nucleotides comprising one or morecomplete or incomplete 3′ UTRs. The flanking region 106 may alsocomprise a 3′ tailing sequence 110 and a 3′UTR 120.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a stop codon. According to the present invention, multipleserial stop codons may also be used. In one embodiment, the operationregion of the present invention may comprise two stop codons. The firststop codon may be “TGA” and the second stop codon may be selected fromthe group consisting of “TAA,” “TGA” and “TAG.”

Turning to FIG. 2 , the 3′UTR 120 of the second flanking region 106 maycomprise one or more sensor sequences 130. These sensor sequences asdiscussed herein operate as pseudo-receptors (or binding sites) forligands of the local microenvironment of the primary construct orpolynucleotide. For example, microRNA binding sites or miRNA seeds maybe used as sensors such that they function as pseudoreceptors for anymicroRNAs present in the environment of the polynucleotide.

As shown in FIG. 3 , FIG. 4 , FIG. 5 and FIG. 6 , the primary construct100 may comprise at least one longevity enhancing sequence 140, such as,but not limited to, a carboxy-terminal peptide sequence, an albuminsequence or an IgG4 sequence may be located in the flanking region 104such as, but not limited to, the 5′UTR as shown in FIG. 3 , the 3′UTR120 as shown in FIG. 4 , the first region of linked nucleosides 102,both the flanking region 104 and 3′UTR 120 as shown in FIG. 5 , or inthe flanking region 104, the first region of linked nucleosides 102 andthe 3′UTR 120 as shown in FIG. 6 . For example, the primary construct100 may include at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10 or morethan 10 longevity enhancing sequences 140.

Generally, the shortest length of the first region of the primaryconstruct of the present invention can be the length of a nucleic acidsequence that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

Generally, the length of the first region encoding the polypeptide ofinterest of the present invention is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, the “first region” may be referred to as a “coding region”or “region encoding” or simply the “first region.”

In some embodiments, the polynucleotide polynucleotide, primaryconstruct, or mmRNA includes from about 30 to about 100,000 nucleotides(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500,from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000,from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000,from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000,from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000,from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000,from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from2,000 to 70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA binding protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofpolyA binding protein. PolyA binding protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to astart and/or stop codon, one or more signal and/or restrictionsequences.

Cyclic Polynucleotides

According to the present invention, a primary construct or mmRNA may becyclized, or concatemerized, to generate a translation competentmolecule to assist interactions between poly-A binding proteins and5′-end binding proteins. The mechanism of cyclization orconcatemerization may occur through at least 3 different routes: 1)chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed5′-/3′-linkage may be intramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acid maycontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NHS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split oligonucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

mmRNA Multimers

According to the present invention, multiple distinct polynucleotides,primary constructs or mmRNA may be linked together through the 3′-endusing nucleotides which are modified at the 3′-terminus. Chemicalconjugation may be used to control the stoichiometry of delivery intocells. For example, the glyoxylate cycle enzymes, isocitrate lyase andmalate synthase, may be supplied into HepG2 cells at a 1:1 ratio toalter cellular fatty acid metabolism. This ratio may be controlled bychemically linking polynucleotides, primary constructs or mmRNA using a3′-azido terminated nucleotide on one polynucleotide, primary constructor mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide onthe opposite polynucleotide, primary construct or mmRNA species. Themodified nucleotide is added post-transcriptionally using terminaltransferase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. After the addition of the 3′-modifiednucleotide, the two polynucleotide, primary construct or mmRNA speciesmay be combined in an aqueous solution, in the presence or absence ofcopper, to form a new covalent linkage via a click chemistry mechanismas described in the literature.

In another example, more than two polynucleotides may be linked togetherusing a functionalized linker molecule. For example, a functionalizedsaccharide molecule may be chemically modified to contain multiplechemical reactive groups (SH—, NH₂—, N₃, etc. . . . ) to react with thecognate moiety on a 3′-functionalized polynucleotide molecule (i.e., a3′-maleimide ester, 3′-NHS-ester, alkynyl). The number of reactivegroups on the modified saccharide can be controlled in a stoichiometricfashion to directly control the stoichiometric ratio of conjugatedpolynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations

In order to further enhance protein production, polynucleotide, primaryconstructs or mmRNA of the present invention can be designed to beconjugated to other polynucleotides, polypeptides, dyes, intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases, proteins, e.g., glycoproteins, orpeptides, e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell, hormones and hormonereceptors, non-peptidic species, such as lipids, lectins, carbohydrates,vitamins, cofactors, or a drug.

Conjugation may result in increased stability and/or half-life and maybe particularly useful in targeting the polynucleotides, primaryconstructs or mmRNA to specific sites in the cell, tissue or organism.

According to the present invention, the polynucleotide mmRNA or primaryconstructs may be administered with, or further encode one or more ofRNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisenseRNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helixformation, aptamers or vectors, and the like.

Bifunctional mmRNA

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional primary constructs or bifunctional mmRNA). As thename implies, bifunctional polynucleotides are those having or capableof at least two functions. These molecules may also by convention bereferred to as multi-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of apolynucleotide and another molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides

As described herein, provided are polynucleotides and primary constructshaving sequences that are partially or substantially not translatable,e.g., having a noncoding region. Such noncoding region may be the “firstregion” of the primary construct. Alternatively, the noncoding regionmay be a region other than the first region. Such molecules aregenerally not translated, but can exert an effect on protein productionby one or more of binding to and sequestering one or more translationalmachinery components such as a ribosomal protein or a transfer RNA(tRNA), thereby effectively reducing protein expression in the cell ormodulating one or more pathways or cascades in a cell which in turnalters protein levels. The polynucleotide and/or primary construct maycontain or encode one or more long noncoding RNA (lncRNA, or lincRNA) orportion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA),small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Polypeptides of Interest

According to the present invention, the primary construct is designed toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “polypeptides ofinterest” refers to any polypeptide which is selected to be encoded inthe primary construct of the present invention. As used herein,“polypeptide” means a polymer of amino acid residues (natural orunnatural) linked together most often by peptide bonds. The term, asused herein, refers to proteins, polypeptides, and peptides of any size,structure, or function. In some instances the polypeptide encoded issmaller than about 50 amino acids and the polypeptide is then termed apeptide. If the polypeptide is a peptide, it will be at least about 2,3, 4, or at least 5 amino acid residues long. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidessuch as antibodies or insulin and may be associated or linked. Mostcommonly disulfide linkages are found in multichain polypeptides. Theterm polypeptide may also apply to amino acid polymers in which one ormore amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, polynucleotides encoding polypeptides containing substitutions,insertions and/or additions, deletions and covalent modifications withrespect to reference sequences, in particular the polypeptide sequencesdisclosed herein, are included within the scope of this invention. Forexample, sequence tags or amino acids, such as one or more lysines, canbe added to the peptide sequences of the invention (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of the aminoacid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the primary construct ormmRNA of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning an polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Polypeptides

The primary constructs or mmRNA of the present invention may be designedto encode polypeptides of interest such as peptides and proteins.

In one embodiment primary constructs or mmRNA of the present inventionmay encode variant polypeptides which have a certain identity with areference polypeptide sequence. As used herein, a “reference polypeptidesequence” refers to a starting polypeptide sequence. Reference sequencesmay be wild type sequences or any sequence to which reference is made inthe design of another sequence. A “reference polypeptide sequence” may,e.g., be any one of the protein sequences known in the art. Non-limitingexamples of protein sequences which may be a reference polypeptidesequence are listed in Table 6 of U.S. Provisional Patent ApplicationNo. 61/618,862, filed Apr. 2, 2012, entitled Modified Polynucleotidesfor the Production of Biologics; U.S. Provisional Patent Application No.61/681,645, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Biologics; U.S. Provisional Patent Application No.61/737,130, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Biologics; International Patent Application No.PCT/US2013/030062, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Biologics and Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,866, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofAntibodies; U.S. Provisional Patent Application No. 61/681,647, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofAntibodies; U.S. Provisional Patent Application No. 61/737,134, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofAntibodies; U.S. Provisional Patent Application No. 61/618,868, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/681,648, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/737,135, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/618,873, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins; U.S. Provisional Patent Application No. 61/681,650,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins; U.S. Provisional Patent Application No.61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Secreted Proteins; International Patent ApplicationNo. PCT/US2013/030064, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Secreted Proteins; U.S.Provisional Patent Application No. 61/618,878, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/681,654, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins; U.S. Provisional Patent Application No. 61/737,152,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Plasma Membrane Proteins; International Patent ApplicationNo. PCT/US2013/030059, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Membrane Proteins; U.S.Provisional Patent Application No. 61/618,885, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Cytoplasmic andCytoskeletal Proteins; U.S. Provisional Patent Application No.61/681,658, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Cytoplasmic and Cytoskeletal Proteins; U.S.Provisional Patent Application No. 61/737,155, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Cytoplasmic andCytoskeletal Proteins; International Patent Application No.PCT/US2013/030066, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cytoplasmic and Cytoskeletal Proteins; U.S.Provisional Patent Application No. 61/618,896, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of IntracellularMembrane Bound Proteins; U.S. Provisional Patent Application No.61/668,157, filed Jul. 5, 2012, entitled Modified Polynucleotides forthe Production of Intracellular Membrane Bound Proteins; U.S.Provisional Patent Application No. 61/681,661, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of IntracellularMembrane Bound Proteins; U.S. Provisional Patent Application No.61/737,160, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Intracellular Membrane Bound Proteins; U.S.Provisional Patent Application No. 61/618,911, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins; U.S. Provisional Patent Application No. 61/681,667, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofNuclear Proteins; U.S. Provisional Patent Application No. 61/737,168,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Nuclear Proteins; International Patent Application No.PCT/US2013/030067, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Nuclear Proteins; U.S. Provisional PatentApplication No. 61/618,922, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/681,675, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/737,174, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; International PatentApplication No. PCT/US2013/030060, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/618,935, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,687, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,184, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; International Patent Application No. PCT/US2013/030061, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; in Tables 6 and 7 of U.S.Provisional Patent Application No. 61/681,720, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; U.S. Provisional Patent Application No.61/737,213, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Cosmetic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/681,742, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Oncology-Related Proteinsand Peptides; International Application No. PCT/US2013/030070, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofOncology-Related Proteins and Peptides; in Tables 6, 178 and 179 ofco-pending International Application No. PCT/US2013/030068, filed Mar.9, 2013, entitled Modified Polynucleotides for the Production ofCosmetic Proteins and Peptides; in Tables 6, 28 and 29 of U.S.Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; in Tables 6, 56 and 57 of U.S. Provisional PatentApplication No. 61/681,649, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides;in Tables 6, 186 and 187 of U.S. Provisional Patent Application No.61/737,139, filed Dec. 14, 2012, Modified Polynucleotides for theProduction of Therapeutic Proteins and Peptides; in Tables 6, 185 and186 of co-pending International Application No PCT/US2013/030063, filedMar. 9, 2013, entitled Modified Polynucleotides; in Table 6 ofco-pending International Application No. PCT/US2013/031821, filed Mar.15, 2013, entitled In Vivo Production of Proteins, the contents of eachof which are herein incorporated by reference in their entireties.

In another embodiment, the polypeptide of interest is the C-terminalfragment (amino acid 180-251) of Fibroblast Growth Factor 23 (FGF23)(amino acid sequence of C-terminal fragment shown in SEQ ID NO: 4).FGF23 is derived from osteocytes and it is believed that FGF23 acts toregulate kidney function and osteogenesis in a klotho dependent fashion(see e.g., Medici et al. FGF-23-Klotho signaling stimulatesproliferation and prevents vitamin D-induced apoptosis. J. Cell Biol.2008: 182(3) 459-465; and Goetz et al. Isolated C-terminal tail of FGF23alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complexformation. PNAS 2010: 107(1) 407-412; each of which is hereinincorporated by reference in its entirety). The C-terminal fragment ofFGF23 could be used as an inhibitor of renal phosphate reabsorption andthe C-terminal fragment could be used to stimulate mitogenic and cellsurvival pathways to prevent atrophy of tissues caused by excessiveVitamin D. Further, the C-terminal fragment of FGF23 can act as acompetitive inhibitor of full length FGF23 to promote renal phosphatereabsorption and correct other diseases caused by the hyperactivity ofFGF23.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into thepolynucleotides, primary constructs and/or mmRNA of the presentinvention to enhance the stability of the molecule. The specificregulatory features can also be incorporated to ensure controlleddown-regulation of the transcript in case they are misdirected toundesired organs sites. One or more carboxy-terminal peptides, albuminor IgG4 sequences described herein may be located before and/or after anuntranslated region of the polynucleotides, primary constructs and/ormmRNA described herein.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG (SEQ ID NO: 51), where R is a purine (adenine or guanine)three bases upstream of the start codon (AUG), which is followed byanother ‘G’. 5′UTR also have been known to form secondary structureswhich are involved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides, primary constructs or mmRNAof the invention. For example, introduction of 5′ UTR of liver-expressedmRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E,transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could beused to enhance expression of a nucleic acid molecule, such as a mmRNA,in hepatic cell lines or liver. Likewise, use of 5′ UTR from othertissue-specific mRNA to improve expression in that tissue ispossible—for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the polynucleotides, primaryconstructs or mmRNA of the invention. Incorporation of intronicsequences may increase protein production as well as mRNA levels.

3′ UTR and the AU Rich Elements

3′UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-α. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides, primaryconstructs or mmRNA of the invention. When engineering specificpolynucleotides, primary constructs or mmRNA, one or more copies of anARE can be introduced to make polynucleotides, primary constructs ormmRNA of the invention less stable and thereby curtail translation anddecrease production of the resultant protein. Likewise, AREs can beidentified and removed or mutated to increase the intracellularstability and thus increase translation and production of the resultantprotein. Transfection experiments can be conducted in relevant celllines, using polynucleotides, primary constructs or mmRNA of theinvention and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and7 days post-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The polynucleotides, primary constructs or mmRNA of theinvention may comprise one or more microRNA target sequences, microRNAsequences, microRNA binding sites, or microRNA seeds. Such sequences maycorrespond to any known microRNA such as those taught in US PublicationUS2005/0261218 and US Publication US2005/0059005, or those listed inTable 7 of application U.S. Ser. No. 61/758,921 filed Jan. 31, 2013, thecontents of which are incorporated herein by reference in theirentirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of polynucleotides,primary constructs or mmRNA of the invention one can target the moleculefor degradation or reduced translation, provided the microRNA inquestion is available. This process will reduce the hazard of off targeteffects upon nucleic acid molecule delivery. Identification of microRNA,microRNA target regions, and their expression patterns and role inbiology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129:1401-1414).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′UTR ofthe polynucleotides, primary constructs or mmRNA. Introduction of one ormultiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of apolynucleotides, primary constructs or mmRNA.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides, primary constructsor mmRNA of the present invention, microRNA binding sites can beengineered out of (i.e. removed from) sequences in which they naturallyoccur in order to increase protein expression in specific tissues. Forexample, miR-122 binding sites may be removed to improve proteinexpression in the liver. Regulation of expression in multiple tissuescan be accomplished through introduction or removal or one or severalmicroRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176). In the polynucleotides,primary constructs or mmRNA of the invention, binding sites formicroRNAs that are involved in such processes may be removed orintroduced, in order to tailor the expression of the polynucleotides,primary constructs or mmRNA expression to biologically relevant celltypes or to the context of relevant biological processes.

Lastly, through an understanding of the expression patterns of microRNAin different cell types, polynucleotides, primary constructs or mmRNAcan be engineered for more targeted expression in specific cell types oronly under specific biological conditions. Through introduction oftissue-specific microRNA binding sites, polynucleotides, primaryconstructs or mmRNA could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered polynucleotides, primary constructs or mmRNA and proteinproduction can be assayed at various time points post-transfection. Forexample, cells can be transfected with different microRNA bindingsite-engineering polynucleotides, primary constructs or mmRNA and byusing an ELISA kit to the relevant protein and assaying protein producedat 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. Invivo experiments can also be conducted using microRNA-bindingsite-engineered molecules to examine changes in tissue-specificexpression of formulated polynucleotides, primary constructs or mmRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the polynucleotides, primary constructs, and mmRNA ofthe present invention may generate a non-hydrolyzable cap structurepreventing decapping and thus increasing mRNA half-life. Because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, modified nucleotides may be used during the capping reaction.For example, a Vaccinia Capping Enzyme from New England Biolabs(Ipswich, Mass.) may be used with α-thio-guanosine nucleotides accordingto the manufacturer's instructions to create a phosphorothioate linkagein the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may beused such as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methylated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptsremain uncapped. This, as well as the structural differences of a capanalog from an endogenous 5′-cap structures of nucleic acids produced bythe endogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Polynucleotides, primary constructs and mmRNA of the invention may alsobe capped post-transcriptionally, using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′cap structures of the present invention arethose which, among other things, have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNAand a guanine cap nucleotide wherein the cap guanine contains an N7methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl. Such a structure is termed the Cap1 structure. This capresults in a higher translational-competency and cellular stability anda reduced activation of cellular pro-inflammatory cytokines, ascompared, e.g., to other 5′cap analog structures known in the art. Capstructures include 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp(cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

Because the polynucleotides, primary constructs or mmRNA may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the polynucleotides, primary constructs or mmRNA may becapped. This is in contrast to ˜80% when a cap analog is linked to anmRNA in the course of an in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can beengineered and inserted in the 3′ UTR of the polynucleotides, primaryconstructs or mmRNA of the invention and can stimulate the translationof the construct in vitro and in vivo. Transfection experiments can beconducted in relevant cell lines at and protein production can beassayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7post-transfection.

IRES Sequences

Further, provided are polynucleotides, primary constructs or mmRNA whichmay contain an internal ribosome entry site (IRES). First identified asa feature Picorna virus RNA, IRES plays an important role in initiatingprotein synthesis in absence of the 5′ cap structure. An IRES may act asthe sole ribosome binding site, or may serve as one of multiple ribosomebinding sites of an mRNA. Polynucleotides, primary constructs or mmRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic nucleic acid molecules”). Whenpolynucleotides, primary constructs or mmRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between 100 and 250residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the polynucleotides, primary constructs or mmRNA of thepresent invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the polynucleotides,primary construct, or mmRNA includes from about 30 to about 3,000nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000,from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall polynucleotides, primary constructs or mmRNA. This designmay be based on the length of the coding region, the length of aparticular feature or region (such as the first or flanking regions), orbased on the length of the ultimate product expressed from thepolynucleotides, primary constructs or mmRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotides, primaryconstructs or mmRNA or feature thereof. The poly-A tail may also bedesigned as a fraction of polynucleotides, primary constructs or mmRNAto which it belongs. In this context, the poly-A tail may be 10, 20, 30,40, 50, 60, 70, 80, or 90% or more of the total length of the constructor the total length of the construct minus the poly-A tail. Further,engineered binding sites and conjugation of polynucleotides, primaryconstructs or mmRNA for Poly-A binding protein may enhance expression.

Additionally, multiple distinct polynucleotides, primary constructs ormmRNA may be linked together to the PABP (Poly-A binding protein)through the 3′-end using modified nucleotides at the 3′-terminus of thepoly-A tail. Transfection experiments can be conducted in relevant celllines and protein production can be assayed by ELISA at 12 hr, 24 hr, 48hr, 72 hr and day 7 post-transfection.

In one embodiment, the polynucleotides and primary constructs of thepresent invention are designed to include a polyA-G Quartet. TheG-quartet is a cyclic hydrogen bonded array of four guanine nucleotidesthat can be formed by G-rich sequences in both DNA and RNA. In thisembodiment, the G-quartet is incorporated at the end of the poly-A tail.The resultant mmRNA construct is assayed for stability, proteinproduction and other parameters including half-life at various timepoints. It has been discovered that the polyA-G quartet results inprotein production equivalent to at least 75% of that seen using apoly-A tail of 120 nucleotides alone.

Quantification

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be quantified in exosomes derived from one ormore bodily fluid. As used herein “bodily fluids” include peripheralblood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.Alternatively, exosomes may be retrieved from an organ selected from thegroup consisting of lung, heart, pancreas, stomach, intestine, bladder,kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus,liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of polynucleotides, primaryconstruct or mmRNA may be an expression level, presence, absence,truncation or alteration of the administered construct. It isadvantageous to correlate the level with one or more clinical phenotypesor with an assay for a human disease biomarker. The assay may beperformed using construct specific probes, cytometry, qRT-PCR, real-timePCR, PCR, flow cytometry, electrophoresis, mass spectrometry, orcombinations thereof while the exosomes may be isolated usingimmunohistochemical methods such as enzyme linked immunosorbent assay(ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides, primary constructs or mmRNAremaining or delivered. This is possible because the polynucleotides,primary constructs or mmRNA of the present invention differ from theendogenous forms due to the structural and/or chemical modifications.

II. Design and Synthesis of Polynucleotides

Polynucleotides, primary constructs or mmRNA for use in accordance withthe invention may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,which is generally termed in vitro transcription (IVT) or enzymatic orchemical cleavage of a longer precursor, etc. Methods of synthesizingRNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:TRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The process of design and synthesis of the primary constructs of theinvention generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies) and/or DNA2.0 (Menlo Park Calif.). In one embodiment, theORF sequence is optimized using optimization algorithms. Codon optionsfor each amino acid are given in Table 2.

TABLE 2 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the primary construct and may flankthe ORF as a first or second flanking region. The flanking regions maybe incorporated into the primary construct before and/or afteroptimization of the ORF. It is not required that a primary constructcontain both a 5′ and 3′ flanking region. Examples of such featuresinclude, but are not limited to, untranslated regions (UTRs), Kozaksequences, an oligo(dT) sequence, and detectable tags and may includemultiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail.

Tables 2 and 3 of co-pending International Application No.PCT/US2013/030062, filed Mar. 9, 2013 the contenct of which are hereinincorporated by reference in its entirety, provide a listing ofexemplary UTRs which may be utilized in the primary construct of thepresent invention as flanking regions. Variants of 5′ UTRs may beutilized wherein one or more nucleotides are added or removed to thetermini, including A, T, C or G.

It should be understood that those listed in the previous tables areexamples and that any UTR from any gene may be incorporated into therespective first or second flanking region of the primary construct.Furthermore, multiple wild-type UTRs of any known gene may be utilized.It is also within the scope of the present invention to provideartificial UTRs which are not variants of wild type genes. These UTRs orportions thereof may be placed in the same orientation as in thetranscript from which they were selected or may be altered inorientation or location. Hence a 5′ or 3′ UTR may be inverted,shortened, lengthened, made chimeric with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may have a heterologous UTR. As used herein“heterologous UTRs” are those UTRs which are not naturally found withthe coding region encoded on the same or instant polynucleotide, primaryconstruct or mmRNA. As a non-limiting example, the first flanking regionmay comprise a heterologous UTR. As another non-limiting example, thesecond flanking region may comprise a heterologous UTR. As yet anothernon-limiting example, the first and second flanking regions may eachcomprise a heterologous UTR. The heterologous UTR in the first flankingregion may be derived from the same species or a different species thanthe heterologous UTR in the second flanking region.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may have a heterologous UTR which is notderived from the beta-globin gene. As a non-limiting example, theheterologous UTR may be a 5′UTR and is not derived from the beta-globingene. As another non-limiting example, the heterologous UTR may be a3′UTR and is not derived from the beta-globin gene.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention comprise a heterologous 5′UTR with the provisothat the heterologous 5′UTR is not derived from the beta-globin gene.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention comprise a heterologous 3′UTR with the provisothat the heterologous 3′UTR is not derived from the beta-globin gene.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may have a homologous UTRs. As used herein“homologous UTRs” are those UTRs which are naturally found associatedwith the coding region of the mRNA, such as the wild type UTR. As anon-limiting example, the first flanking region may comprise ahomologous UTR. As another non-limiting example, the second flankingregion may comprise a homologous UTR. As yet another non-limitingexample, the first and second flanking regions may each comprise ahomologous UTR.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may have a heterologous UTR in the firstflanking region and a homologous UTR in the second flanking region.

In another embodiment, the polynucleotides, primary constructs and/ormmRNA of the present invention may have a homologous UTR in the firstflanking region and a heterologous UTR in the second flanking region.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as, but not limited to, AB, ABA,ABAB, ABABA, ABABAB, AAB, AABB, AABBA, AABBAA, AABBAAB, AABBAABB,AABBAABBA, ABB, ABBA, AABBAABBAABB, ABC, ABCA, ABCAB, ABCABC, ABCABCA,ABCABCAB, ABCABCABC, ABCB, ABCBC, ABCBCA, ABCC, ABCCB, ABCCBA, orvariants thereof repeated once, twice, or more than 3 times. In thesepatterns, each letter, A, B, or C represent a different UTR at thenucleotide level. The different UTRs represented in each pattern may bederived from the same species or they may be derived from differentspecies.

In one embodiment, the flanking regions may comprise patterned UTRs. Inone embodiment, the first flanking region and the second flanking regionmay each comprise a patterned UTR. The pattern for each UTR may be thesame or different. As a non-limiting example, the patterned UTR in firstflanking region is different than the patterned UTR in the secondflanking region. As another non-limiting example, the patterned UTR inthe first flanking region and the second flanking region may be thesame.

In one embodiment, the flanking regions may comprise patterned UTRsderived from the same species. As a non-limiting example, the patternedUTR in the first flanking region may be derived from the same species asthe patterned UTR in the second flanking region, but the patterned UTRin the first flanking region is different from the patterned UTR in thesecond flanking region.

In one embodiment, the first flanking region may comprise a patternedUTR derived from a first species and the second flanking region maycomprise a patterned UTR derived from a second species.

In another embodiment, the flanking regions may comprise patterned UTRsderived from different species.

In one embodiment, the patterned UTR may comprise heterologous andhomologous UTRs. As a non-limiting example, the first flanking regionmay comprise heterologous UTRs and the second flanking region maycomprise homologous UTRs.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new chimeric primary transcript. As used herein, a “family ofproteins” is used in the broadest sense to refer to a group of two ormore polypeptides of interest which share at least one function,structure, feature, localization, origin, or expression pattern.

After optimization (if desired), the primary construct components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized construct may be reconstituted and transformed into chemicallycompetent E. coli, yeast, Neurospora, maize, Drosophila, etc. where highcopy plasmid-like or chromosome structures occur by methods describedherein.

Stop Codons

In one embodiment, the primary constructs of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA.

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionwhich was conducted. The linearized plasmid is then used to generatecDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 4 of InternationalApplication No. PCT/US2013/030062, filed Mar. 9, 2013, the contents ofwhich are herein incorporated by reference in its entirety, provides alisting of primers and probes that may be usefully in the PCR reactionsof the present invention. is a listing of primers and probes that may beuseful in the PCR reactions of the present invention. It should beunderstood that the listing is not exhaustive and that primer-probedesign for any amplification is within the skill of those in the art.Probes may also contain chemically modified bases to increasebase-pairing fidelity to the target molecule and base-pairing strength.Such modifications may include 5-methyl-Cytidine, 2,6-di-amino-purine,2′-fluoro, phosphoro-thioate, or locked nucleic acids.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

mRNA Production

The process of polynucleotide production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andcapping and/or tailing reactions.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to be incorporated into modified nucleic acids.

RNA Polymerases

Any number of RNA polymerases or variants may be used in the design ofthe constructs of the present invention.

RNA polymerases may be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase may encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L6991, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants may encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one embodiment, the primary construct may be designed to berecognized by the wild type or variant RNA polymerases. In doing so, theprimary construct may be modified to contain sites or regions ofsequence changes from the wild type or parent primary construct.

In one embodiment, the primary construct may be designed to include atleast one substitution and/or insertion upstream of an RNA polymerasebinding or recognition site, downstream of the RNA polymerase binding orrecognition site, upstream of the TATA box sequence, downstream of theTATA box sequence of the primary construct but upstream of the codingregion of the primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least one region and/or string of nucleotides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleic acid may cause a silent mutation of the nucleic acidsequence or may cause a mutation in the amino acid sequence.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Theprimary construct may include, but is not limited to, at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7 orat least 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in the primaryconstruct may be substituted with adenine, cytosine, thymine, or any ofthe nucleotides described herein. In another non-limiting example thesubstitution of guanine bases in the primary construct may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; herein incorporated by reference in itsentirety). As a non-limiting example, at least 5 nucleotides may beinserted at 1 location downstream of the transcription start site butupstream of the start codon and the at least 5 nucleotides may be thesame base type.

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The primary construct or mmRNA may also undergo capping and/or tailingreactions. A capping reaction may be performed by methods known in theart to add a 5′ cap to the 5′ end of the primary construct. Methods forcapping include, but are not limited to, using a Vaccinia Capping enzyme(New England Biolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the primary construct generated from cDNA does notinclude a poly-T, it may be beneficial to perform the poly-A-tailingreaction before the primary construct is cleaned.

Purification

Primary construct or mmRNA purification may include, but is not limitedto, mRNA or mmRNA clean-up, quality assurance and quality control. mRNAor mmRNA clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedmRNA or mmRNA” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the mRNA or mmRNA may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

In one embodiment, the mRNA or mmRNA may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA ormmRNA may be analyzed in order to determine if the mRNA or mmRNA may beof proper size, check that no degradation of the mRNA or mmRNA hasoccurred. Degradation of the mRNA and/or mmRNA may be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

Signal Peptides or Proteins

The primary constructs or mmRNA may also encode additional featureswhich facilitate trafficking of the polypeptides to therapeuticallyrelevant sites. One such feature which aids in protein trafficking isthe signal peptide sequence. As used herein, a “signal sequence” or“signal peptide” is a polynucleotide or polypeptide, respectively, whichis from about 9 to 200 nucleotides (3-60 amino acids) in length which isincorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Signal sequences may be selected from any of those listed in Table 3below and those listed in Table 5 of co-pending InternationalApplication No. PCT/US2013/030062, filed Mar. 9, 2013, the contents ofwhich are incorporated herein by reference.

TABLE 3 Signal Peptides SEQ ID Description Sequence NO HSA/KEX2 SignalMKWVSFISLLFLFSSAYSGSLDKR 5 Peptide FGF23 Signal MLGARLRLWVCALCSVCSMSVLR6 Peptide A hGH Signal MATGSRTSLLLAFGLLCLPWLQE 7 Peptide GSA GCSF SignalMAGPATQSPMKLMALQLLLWHS 8 Peptide ALWTVQE

Protein signal sequences which may be incorporated for encoding by thepolynucleotides, primary constructs or mmRNA of the invention includesignal sequences from α-1-antitrypsin, G-CSF, Factor IX, Prolactin,Albumin, HMMSP38, ornithine carbamoyltransferase, Cytochrome C Oxidasesubunit 8A, Type III, bacterial, viral, secretion signals, Vrg-6, PhoA,OmpA, STI, STII, Amylase, Alpha Factor, Endoglucanase V, Secretionsignal, fungal and fibronectin.

In the Table 5 of co-pending International Application No.PCT/US2013/030062, SS is secretion signal and MLS is mitochondrialleader signal. The primary constructs or mmRNA of the present inventionmay be designed to encode any of the signal peptide sequences orfragments or variants thereof. These sequences may be included at thebeginning of the polypeptide coding region, in the middle or at theterminus or alternatively into a flanking region.

Additional signal peptide sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at http://world wide web.signalpeptide.de/orhttp://proline.dot.bic.dot.nus.dot.edu.dot.sg/spdb/. Those described inU.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are also within thescope of the invention and the contents of each are incorporated hereinby reference in their entirety.

Target Selection

According to the present invention, the primary constructs comprise atleast a first region of linked nucleosides encoding at least onepolypeptide of interest. The polypeptides of interest or “targets” orproteins and peptides of the present invention are listed in Table 6 ofU.S. Provisional Patent Application No. 61/618,862, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Biologics;International Patent Application No. PCT/US2013/030062, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Biologicsand Proteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,866, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Antibodies; U.S. ProvisionalPatent Application No. 61/681,647, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins; U.S. Provisional Patent Application No. 61/681,650, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins; U.S. Provisional Patent Application No. 61/737,147,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins; International Patent Application No.PCT/US2013/030064, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Secreted Proteins; U.S. Provisional PatentApplication No. 61/618,878, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins; U.S.Provisional Patent Application No. 61/681,654, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/737,152, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins; International Patent Application No.PCT/US2013/030059, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Membrane Proteins; U.S. Provisional PatentApplication No. 61/618,885, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; U.S. Provisional Patent Application No. 61/681,658, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/737,155, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; International Patent Application No. PCT/US2013/030066, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins; U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins; U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins; International Patent Application No. PCT/US2013/030067, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofNuclear Proteins; U.S. Provisional Patent Application No. 61/618,922,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Proteins; U.S. Provisional Patent Application No. 61/681,675, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins; International Patent Application No. PCT/US2013/030060, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; International Patent ApplicationNo. PCT/US2013/030061, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,945, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,696, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,191, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,953, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,704, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,203, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; in Tables 6 and 7 of U.S. Provisional Patent Application No.61/681,720, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Cosmetic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/737,213, filed Dec. 14, 2012, entitledModified Polynucleotides for the Production of Cosmetic Proteins andPeptides; U.S. Provisional Patent Application No. 61/681,742, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofOncology-Related Proteins and Peptides; International Application No.PCT/US2013/030070, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Oncology-Related Proteins and Peptides; in Tables6, 178 and 179 of co-pending International Application No.PCT/US2013/030068, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cosmetic Proteins and Peptides; in Tables 6, 28and 29 of U.S. Provisional Patent Application No. 61/618,870, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofTherapeutic Proteins and Peptides; in Tables 6, 56 and 57 of U.S.Provisional Patent Application No. 61/681,649, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; in Tables 6, 186 and 187 of U.S. ProvisionalPatent Application No. 61/737,139, filed Dec. 14, 2012, ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides;in Tables 6, 185 and 186 of co-pending International Application NoPCT/US2013/030063, filed Mar. 9, 2013, entitled ModifiedPolynucleotides; in Table 6 of co-pending International Application No.PCT/US2013/031821, filed Mar. 15, 2013, entitled In Vivo Production ofProteins, the contents of each of which are herein incorporated byreference in their entireties.

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). In oneembodiment, the primary constructs and the mmRNA of the presentinvention may be engineered such that the primary construct or mmRNAcontains at least one encoded protein cleavage signal. The encodedprotein cleavage signal may be located before the start codon, after thestart codon, before the coding region, within the coding region such as,but not limited to, half way in the coding region, between the startcodon and the half way point, between the half way point and the stopcodon, after the coding region, before the stop codon, between two stopcodons, after the stop codon and combinations thereof.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include at least one encoded protein cleavage signalcontaining at least one protein cleavage site. The encoded proteincleavage signal may include, but is not limited to, a proproteinconvertase (or prohormone convertase), thrombin and/or Factor Xa proteincleavage signal. One of skill in the art may use Table 2 above or otherknown methods to determine the appropriate encoded protein cleavagesignal to include in the primary constructs or mmRNA of the presentinvention. In one embodiment, the polypeptides of the present inventioninclude at least one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite with the proviso that the primary construct or mmRNA does notencode GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include more than one coding region. Where multiple codingregions are present in the primary construct or mmRNA of the presentinvention, the multiple coding regions may be separated by encodedprotein cleavage sites. As a non-limiting example, the primary constructor mmRNA may be signed in an ordered pattern. On such pattern followsAXBY form where A and B are coding regions which may be the same ordifferent coding regions and/or may encode the same or differentpolypeptides, and X and Y are encoded protein cleavage signals which mayencode the same or different protein cleavage signals. A second suchpattern follows the form AXYBZ where A and B are coding regions whichmay be the same or different coding regions and/or may encode the sameor different polypeptides, and X, Y and Z are encoded protein cleavagesignals which may encode the same or different protein cleavage signals.A third pattern follows the form ABXCY where A, B and C are codingregions which may be the same or different coding regions and/or mayencode the same or different polypeptides, and X and Y are encodedprotein cleavage signals which may encode the same or different proteincleavage signals.

In on embodiment, the polypeptides, primary constructs and mmRNA canalso contain sequences that encode protein cleavage sites so that thepolypeptides, primary constructs and mmRNA can be released from acarrier region or a fusion partner by treatment with a specific proteasefor said protein cleavage site.

III. Modifications

Herein, in a polynucleotide (such as a primary construct or a mRNAmolecule), the terms “modification” or, as appropriate, “modified” referto modification with respect to A, G, U or C ribonucleotides. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties. In apolypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids.

The modifications may be various distinct modifications. In someembodiments, the coding region, the flanking regions and/or the terminalregions may contain one, two, or more (optionally different) nucleosideor nucleotide modifications. In some embodiments, a modifiedpolynucleotide, primary construct, or mmRNA introduced to a cell mayexhibit reduced degradation in the cell, as compared to an unmodifiedpolynucleotide, primary construct, or mmRNA.

The polynucleotides, primary constructs, and mmRNA can include anyuseful modification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleoside linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

As described herein, in some embodiments, the polynucleotides, primaryconstructs, and mmRNA of the invention do not substantially induce aninnate immune response of a cell into which the mRNA is introduced.Featues of an induced innate immune response include 1) increasedexpression of pro-inflammatory cytokines, 2) activation of intracellularPRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in proteintranslation. In other embodiments, an immune response is induced.

In certain embodiments, it may desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example,degradation of a modified nucleic acid molecule may be preferable ifprecise timing of protein production is desired. Thus, in someembodiments, the invention provides a modified nucleic acid moleculecontaining a degradation domain, which is capable of being acted on in adirected manner within a cell.

In another aspect, the present disclosure provides polynucleotidescomprising a nucleoside or nucleotide that can disrupt the binding of amajor groove interacting, e.g. binding, partner with the polynucleotide(e.g., where the modified nucleotide has decreased binding affinity tomajor groove interacting partner, as compared to an unmodifiednucleotide).

The polynucleotides, primary constructs, and mmRNA can optionallyinclude other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAsthat induce triple helix formation, aptamers, vectors, etc.). In someembodiments, the polynucleotides, primary constructs, or mmRNA mayinclude one or more messenger RNAs (mRNAs) and one or more modifiednucleoside or nucleotides (e.g., mmRNA molecules). Details for thesepolynucleotides, primary constructs, and mmRNA follow.

Polynucleotides and Primary Constructs

The polynucleotides, primary constructs, and mmRNA of the inventionincludes a first region of linked nucleosides encoding a polypeptide ofinterest, a first flanking region located at the 5′ terminus of thefirst region, and a second flanking region located at the 3′ terminus ofthe first region.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having any base, sugar,backbone, building block or other structure or formula, including butnot limited to those of Formulas I through IX or any substructuresthereof as described in International Application No. PCT/US2012/58519,the contents of which are incorporated herein by reference in theirentirety. Such structures include modifications to the sugar,nucleobase, internucleoside linkage, or combinations thereof.

Combinations of chemical modifications include those taught in includingbut not limited to those described in International Application No.PCT/US2012/58519, the contents of which are incorporated herein byreference in their entirety.

The synthesis of polynucleotides, primary constructs or mmRNA of thepresent invention may be according to the methods described inInternational Application No. PCT/US2012/58519, U.S. Provisional PatentApplication No. 61/618,862, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Biologics; U.S. Provisional PatentApplication No. 61/681,645, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Biologics; U.S. Provisional PatentApplication No. 61/737,130, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Biologics; International PatentApplication No. PCT/US2013/030062, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Biologics and Proteins Associatedwith Human Disease; U.S. Provisional Patent Application No. 61/618,866,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Antibodies; U.S. Provisional Patent Application No. 61/681,647, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofAntibodies; U.S. Provisional Patent Application No. 61/737,134, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofAntibodies; U.S. Provisional Patent Application No. 61/618,868, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/681,648, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/737,135, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofVaccines; U.S. Provisional Patent Application No. 61/618,873, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins; U.S. Provisional Patent Application No. 61/681,650,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins; U.S. Provisional Patent Application No.61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Secreted Proteins; International Patent ApplicationNo. PCT/US2013/030064, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Secreted Proteins; U.S.Provisional Patent Application No. 61/618,878, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/681,654, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins; U.S. Provisional Patent Application No. 61/737,152,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Plasma Membrane Proteins; International Patent ApplicationNo. PCT/US2013/030059, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Membrane Proteins; U.S.Provisional Patent Application No. 61/618,885, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Cytoplasmic andCytoskeletal Proteins; U.S. Provisional Patent Application No.61/681,658, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Cytoplasmic and Cytoskeletal Proteins; U.S.Provisional Patent Application No. 61/737,155, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Cytoplasmic andCytoskeletal Proteins; International Patent Application No.PCT/US2013/030066, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cytoplasmic and Cytoskeletal Proteins; U.S.Provisional Patent Application No. 61/618,896, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of IntracellularMembrane Bound Proteins; U.S. Provisional Patent Application No.61/668,157, filed Jul. 5, 2012, entitled Modified Polynucleotides forthe Production of Intracellular Membrane Bound Proteins; U.S.Provisional Patent Application No. 61/681,661, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of IntracellularMembrane Bound Proteins; U.S. Provisional Patent Application No.61/737,160, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Intracellular Membrane Bound Proteins; U.S.Provisional Patent Application No. 61/618,911, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins; U.S. Provisional Patent Application No. 61/681,667, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofNuclear Proteins; U.S. Provisional Patent Application No. 61/737,168,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Nuclear Proteins; International Patent Application No.PCT/US2013/030067, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Nuclear Proteins; U.S. Provisional PatentApplication No. 61/618,922, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/681,675, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/737,174, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins; International PatentApplication No. PCT/US2013/030060, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Proteins; U.S. Provisional PatentApplication No. 61/618,935, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,687, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,184, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; International Patent Application No. PCT/US2013/030061, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,720, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cosmetic Proteins and Peptides;U.S. Provisional Patent Application No. 61/737,213, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; U.S. Provisional Patent Application No.61/681,742, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Oncology-Related Proteins and Peptides; InternationalApplication No. PCT/US2013/030070, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Oncology-Related Proteins andPeptides; International Application No. PCT/US2013/030068, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; U.S. Provisional Patent Application No.61/618,870, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/681,649, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Therapeutic Proteins andPeptides; U.S. Provisional Patent Application No. 61/737,139, filed Dec.14, 2012, Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; International Application No PCT/US2013/030063,filed Mar. 9, 2013, entitled Modified Polynucleotides; InternationalApplication No. PCT/US2013/031821, filed Mar. 15, 2013, entitled In VivoProduction of Proteins, the contents of each of which are hereinincorporated by reference in their entireties, the contents of each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the nucleobase selected from the group consistingof cytosine, guanine, adenine, and uracil.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψW), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mCm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(™⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(™⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetylcytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G+),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), and 2′-O-ribosylguanosine(phosphate) (Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polypeptides, primary constructs, and mmRNA of the invention may ormay not be uniformly modified along the entire length of the molecule.For example, one or more or all types of nucleotide (e.g., purine orpyrimidine, or any one or more or all of A, G, U, C) may or may not beuniformly modified in a polynucleotide of the invention, or in a givenpredetermined sequence region thereof (e.g. one or more of the sequenceregions represented in FIG. 1 ). In some embodiments, all nucleotides Xin a polynucleotide of the invention (or in a given sequence regionthereof) are modified, wherein X may any one of nucleotides A, G, U, C,or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the polynucleotide, primary construct, or mmRNA.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of apolynucleotide, primary construct, or mmRNA such that the function ofthe polynucleotide, primary construct, or mmRNA is not substantiallydecreased. A modification may also be a 5′ or 3′ terminal modification.The polynucleotide, primary construct, or mmRNA may contain from about1% to about 100% modified nucleotides (either in relation to overallnucleotide content, or in relation to one or more types of nucleotide,i.e. any one or more of A, G, U or C) or any intervening percentage(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified pyrimidine (e.g., a modified uracil/uridine/U ormodified cytosine/cytidine/C). In some embodiments, the uracil oruridine (generally: U) in the polynucleotide, primary construct, ormmRNA molecule may be replaced with from about 1% to about 100% of amodified uracil or modified uridine (e.g., from 1% to 20%, from 1% to25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100% of a modified uracil or modifieduridine). The modified uracil or uridine can be replaced by a compoundhaving a single unique structure or by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein).

In some embodiments, the cytosine or cytidine (generally: C) in thepolynucleotide, primary construct, or mmRNA molecule may be replacedwith from about 1% to about 100% of a modified cytosine or modifiedcytidine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1%to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%,from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10%to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100% of a modified cytosine or modified cytidine). The modifiedcytosine or cytidine can be replaced by a compound having a singleunique structure or by a plurality of compounds having differentstructures (e.g., 2, 3, 4 or more unique structures, as describedherein).

In some embodiments, the polynucleotide, primary construct, or mmRNA istranslatable.

Other components of polynucleotides, primary constructs, and mmRNA areoptional, and are beneficial in some embodiments. For example, a 5′untranslated region (UTR) and/or a 3′UTR are provided, wherein either orboth may independently contain one or more different nucleotidemodifications. In such embodiments, nucleotide modifications may also bepresent in the translatable region. Also provided are polynucleotides,primary constructs, and mmRNA containing a Kozak sequence.

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

IV. Pharmaceutical Compositions

Formulation, Administration, Delivery and Dosing

The present invention provides polynucleotides, primary constructs andmmRNA compositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides, primaryconstructs and mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more excipients to: (1) increase stability; (2)increase cell transfection; (3) permit the sustained or delayed release(e.g., from a depot formulation of the polynucleotide, primaryconstruct, or mmRNA); (4) alter the biodistribution (e.g., target thepolynucleotide, primary construct, or mmRNA to specific tissues or celltypes); (5) increase the translation of encoded protein in vivo; and/or(6) alter the release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present invention can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotide, primary construct, or mmRNA (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof. Further, the polynucleotide, primary construct, ormmRNA of the present invention may be formulated using self-assemblednucleic acid nanoparticles.

Accordingly, the formulations of the invention can include one or moreexcipients, each in an amount that together increases the stability ofthe polynucleotide, primary construct, or mmRNA, increases celltransfection by the polynucleotide, primary construct, or mmRNA,increases the expression of polynucleotide, primary construct, or mmRNAencoded protein, and/or alters the release profile of polynucleotide,primary construct, or mmRNA encoded proteins. Further, the primaryconstruct and mmRNA of the present invention may be formulated usingself-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the formulations described herein may contain atleast one mmRNA. As a non-limiting example, the formulations may contain1, 2, 3, 4 or 5 mmRNA. In one embodiment the formulation may containmodified mRNA encoding proteins selected from categories such as,proteins. In one embodiment, the formulation contains at least threemodified mRNA encoding proteins. In one embodiment, the formulationcontains at least five modified mRNA encoding proteins.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdescribed herein may be formulated as described in InternationalApplication No PCT/US2012/069610, filed Dec. 14, 2012, entitled ModifiedNucleoside, Nucleotide, and Nucleic Acid Compositions, the contents ofwhich are herein incorporated by reference in its entirety. Non-limitingexamples of formulations include lipidoids, liposomes, lipoplexes, lipidnanoparticles, peptides, proteins, cells, hyaluronidase, nanoparticlemimics, nanotubes, conjugates, self-assembled nanoparticles, inorganicnanoparticles, semi-conductive nanoparticles, metallic nanoparticles,gels, hydrogels, molded nanoparticles, microparticles, nanojackets andnanoliposomes. The formulation may be administered by any of the methodsdescribed in International Application No PCT/US2012/069610, filed Dec.14, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic AcidCompositions, the contents of which are herein incorporated by referencein its entirety. Non limiting examples of administration methods includeparenteral, injectable, rectal, vaginal, oral, topical, transdermal,depot, pulmonary, intranasal, buccal and ophthalmic administration.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference). The use of a conventional excipient medium may becontemplated within the scope of the present disclosure, except insofaras any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides, primary constructs or mmRNA (see Mahon etal., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med.2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., ProcNatl Acad Sci USA. 2011 108:12996-3001; all of which are incorporatedherein in their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded polynucleotides, primary constructs, or mmRNA.Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, primary construct, or mmRNA, as judged by theproduction of an encoded protein, following the injection of a lipidoidformulation via localized and/or systemic routes of administration.Lipidoid complexes of polynucleotides, primary constructs, or mmRNA canbe administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as particle size (Akinc et al., MolTher. 2009 17:872-879; herein incorporated by reference in itsentirety). As an example, small changes in the anchor chain length ofpoly(ethylene glycol) (PEG) lipids may result in significant effects onin vivo efficacy. Formulations with the different lipidoids, including,but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010)), C12-200 (including derivatives and variants), and MD1,can be tested for in vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670; both of which are herein incorporatedby reference in their entirety. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotide, primary construct, or mmRNA. As an example, formulationswith certain lipidoids, include, but are not limited to, 98N12-5 and maycontain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chainlength). As another example, formulations with certain lipidoids,include, but are not limited to, C12-200 and may contain 50% lipidoid,10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

Combinations of different lipidoids may be used to improve the efficacyof polynucleotide, primary construct, or mmRNA directed proteinproduction as the lipidoids may be able to increase cell transfection bythe polynucleotide, primary construct, or mmRNA; and/or increase thetranslation of encoded protein (see Whitehead et al., Mol. Ther. 2011,19:1688-1694, herein incorporated by reference in its entirety).

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of, thepolynucleotide, primary construct, or mmRNA delivered to subjects.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions ofpolynucleotide, primary construct, or mmRNA include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties.) The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the polynucleotide,primary construct, or mmRNA. As an example a liposome can contain, butis not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline(DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane(DODMA), as described by Jeffs et al. As another example, certainliposome formulations may contain, but are not limited to, 48%cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where thecationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA),DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA),as described by Heyes et al.

In one embodiment, pharmaceutical compostions may include liposomeswhich may be formed to deliver mmRNA which may encode at least oneimmunogen. The mmRNA may be encapsulated by the liposome and/or it maybe contained in an aqueous core which may then be encapsulated by theliposome (see International Pub. Nos. WO2012031046, WO2012031043,WO201203091 and WO2012006378 herein incorporated by reference in theirentireties). In another embodiment, the mmRNA which may encode animmunogen may be formulated in a cationic oil-in-water emulsion wherethe emulsion particle comprises an oil core and a cationic lipid whichcan interact with the mmRNA anchoring the molecule to the emulsionparticle (see International Pub. No. WO2012006380). In yet anotherembodiment, the lipid formulation may include at least cationic lipid, alipid which may enhance transfection and a least one lipid whichcontains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582;herein incorporated by reference in their entireties). In anotherembodiment, the polynucleotides, primary constructs and/or mmRNAencoding an immunogen may be formulated in a lipid vesicle which mayhave crosslinks between functionalized lipid bilayers (see U.S. Pub. No.20120177724, herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid vesicle which may have crosslinks betweenfunctionalized lipid bilayers.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid-polycation complex. The formation of thelipid-polycation complex may be accomplished by methods known in the artand/or as described in U.S. Pub. No. 20120178702, herein incorporated byreference in its entirety. As a non-limiting example, the polycation mayinclude a cationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In another embodiment,the polynucleotides, primary constructs and/or mmRNA may be formulatedin a lipid-polycation complex which may further include a neutral lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176), the liposomeformulation was composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid couldmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated byreference in its entirety).

In some embodiments, the ratio of PEG in the LNP formulations may beincreased or decreased and/or the carbon chain length of the PEG lipidmay be modified from C14 to C18 to alter the pharmacokinetics and/orbiodistribution of the LNP formulations. As a non-limiting example, LNPformulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG ascompared to the cationic lipid, DSPC and cholesterol. In anotherembodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, butnot limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethyleneglycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol). The cationic lipid may be selected from any lipid known in theart such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 andDLin-KC2-DMA.

In one embodiment, the LNP formulations of the polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 3% lipid molar ratio. Inanother embodiment, the LNP formulations polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides, primary constructs and/or mmRNA may include at leastone of the PEGylated lipids described in International Publication No.2012099755, herein incorporated by reference.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (QuietTherapeutics, Israel).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon.

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; herein incorporated by reference in theirentireties). The polymer may encapsulate the nanospecies or partiallyencapsulate the nanospecies. The immunogen may be a recombinant protein,a modified RNA and/or a primary construct described herein. In oneembodiment, the lipid nanoparticle may be formulated for use in avaccine such as, but not limited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; herein incorporated by reference in their entirety). Thetransport of nanoparticles may be determined using rates of permeationand/or fluorescent microscopy techniques including, but not limited to,fluorescence recovery after photobleaching (FRAP) and high resolutionmultiple particle tracking (MPT).

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples of specificpolymers include poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication20100003337; herein incorporated by reference in their entireties). Theco-polymer may be a polymer that is generally regarded as safe (GRAS)and the formation of the lipid nanoparticle may be in such a way that nonew chemical entities are created. For example, the lipid nanoparticlemay comprise poloxamers coating PLGA nanoparticles without forming newchemical entities which are still able to rapidly penetrate human mucus(Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic protein(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin34 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see US Publication 20100215580 and US Publication 20080166414; hereinincorporated by reference in their entireties).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; herein incorporated by reference in their entirety). Thetransport of nanoparticles may be determined using rates of permeationand/or fluorescent microscopy techniques including, but not limited to,fluorescence recovery after photobleaching (FRAP) and high resolutionmultiple particle tracking (MPT).

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayincluding, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples of specificpolymers include poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication20100003337; herein incorporated by reference in their entireties).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic protein(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin34 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see US Publication 20100215580 and US Publication 20080166414; hereinincorporated by reference in their entireties).

The mucus penetrating lipid nanoparticles may comprise at least onepolynucleotide, primary construct, or mmRNA described herein. Thepolynucleotide, primary construct, or mmRNA may be encapsulated in thelipid nanoparticle and/or disposed on the surface of the particle. Thepolynucleotide, primary construct, or mmRNA may be covalently coupled tothe lipid nanoparticle. Formulations of mucus penetrating lipidnanoparticles may comprise a plurality of nanoparticles. Further, theformulations may contain particles which may interact with the mucus andalter the structural and/or adhesive properties of the surrounding mucusto decrease mucoadhesion which may increase the delivery of the mucuspenetrating lipid nanoparticles to the mucosal tissue.

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a lipoplex, such as, without limitation, the ATUPLEX™system, the DACC system, the DBTC system and other siRNA-lipoplextechnology from Silence Therapeutics (London, United Kingdom), STEMFECT™from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) orprotamine-based targeted and non-targeted delivery of nucleic acidsacids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. IntJ Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. MicrovascRes 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide etal. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song etal., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl AcadSci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulationswhich have been shown to bind to apolipoprotein E and promote bindingand uptake of these formulations into hepatocytes in vivo (Akinc et al.Mol Ther. 2010 18:1357-1364; herein incorporated by reference in itsentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714Zhao et al., Expert Opin Drug Deliv.2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan etal., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods MolBiol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer etal., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods MolBiol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037;Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety).

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a solid lipid nanoparticle. A solid lipid nanoparticle(SLN) may be spherical with an average diameter between 10 to 1000 nm.SLN possess a solid lipid core matrix that can solubilize lipophilicmolecules and may be stabilized with surfactants and/or emulsifiers. Ina further embodiment, the lipid nanoparticle may be a self-assemblylipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp1696-1702; herein incorporated by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotide, primary construct, or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the polynucleotide, primary construct, or mmRNA; and/orincrease the translation of encoded protein. One such example involvesthe use of lipid encapsulation to enable the effective systemic deliveryof polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; hereinincorporated by reference in its entirety). The liposomes, lipoplexes,or lipid nanoparticles may also be used to increase the stability of thepolynucleotide, primary construct, or mmRNA.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using natural and/or synthetic polymers. Non-limitingexamples of polymers which may be used for delivery include, but are notlimited to, Dynamic POLYCONJUGATE™ formulations from MIRUS® Bio(Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX™ polymerformulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™(Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical(San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals(Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA)polymers. RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers(Arrowhead Research Corporation, Pasadena, Calif.) and pH responsiveco-block polymers such as, but not limited to, PHASERX™ (Seattle,Wash.).

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887). This particular approach is a multicomponent polymersystem whose key features include a membrane-active polymer to whichnucleic acid, in this case siRNA, is covalently coupled via a disulfidebond and where both PEG (for charge masking) and N-acetylgalactosamine(for hepatocyte targeting) groups are linked via pH-sensitive bonds(Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887). Onbinding to the hepatocyte and entry into the endosome, the polymercomplex disassembles in the low-pH environment, with the polymerexposing its positive charge, leading to endosomal escape andcytoplasmic release of the siRNA from the polymer. Through replacementof the N-acetylgalactosamine group with a mannose group, it was shownone could alter targeting from asialoglycoprotein receptor-expressinghepatocytes to sinusoidal endothelium and Kupffer cells. Another polymerapproach involves using transferrin-targeted cyclodextrin-containingpolycation nanoparticles. These nanoparticles have demonstrated targetedsilencing of the EWS-FLI1 gene product in transferrinreceptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al.,Cancer Res. 2005 65: 8984-8982) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21). Both of these deliverystrategies incorporate rational approaches using both targeted deliveryand endosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotide, primary construct, or mmRNA (e.g., followingintramuscular or subcutaneous injection). The altered release profilefor the polynucleotide, primary construct, or mmRNA can result in, forexample, translation of an encoded protein over an extended period oftime. The polymer formulation may also be used to increase the stabilityof the polynucleotide, primary construct, or mmRNA. Biodegradablepolymers have been previously used to protect nucleic acids other thanmmRNA from degradation and been shown to result in sustained release ofpayloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; herein incorporated by reference in its entirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.). TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic ineraction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; herein incorporated by reference in itsentirety).

The mmRNA of the invention may be formulated with or in a polymericcompound. The polymer may include at least one polymer such as, but notlimited to, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG graftedto PLL, cationic lipopolymer, biodegradable cationic lipopolymer,polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), apolyamine derivative, a modified poloxamer, a biodegradable polymer,biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, linearbiodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA),biodegradable cross-linked cationic multi-block copolymers orcombinations thereof.

As a non-limiting example, the mmRNA of the invention may be formulatedwith the polymeric compound of PEG grafted with PLL as described in U.S.Pat. No. 6,177,274 herein incorporated by reference in its entirety. Theformulation may be used for transfecting cells in vitro or for in vivodelivery of the mmRNA. In another example, the mmRNA may be suspended ina solution or medium with a cationic polymer, in a dry pharmaceuticalcomposition or in a solution that is capable of being dried as describedin U.S. Pub. Nos. 20090042829 and 20090042825 each of which are hereinincorporated by reference in their entireties.

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the mmRNA and the polyamine derivative describedin U.S. Pub. No. 20100260817 (the contents of which are incorporatedherein by reference in its entirety.

For example, the mmRNA of the invention may be formulated in apharmaceutical compound including a poly(alkylene imine), abiodegradable cationic lipopolymer, a biodegradable block copolymer, abiodegradable polymer, or a biodegradable random copolymer, abiodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made my methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradabale polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in its entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S.Pub. No. 2012009145 herein incorporated by reference in theirentireties. For example, the multi-block copolymers may be synthesizedusing linear polyethyleneimine (LPEI) blocks which have distinctpatterns as compared to branched polyethyleneimines. Further, thecomposition or pharmaceutical composition may be made by the methodsknown in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 hereinincorporated by reference in their entireties.

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the mmRNA of thepresent invention may be used in a gene delivery composition with thepoloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The polynucleotide, primary construct, and mmRNA of the invention canalso be formulated as a nanoparticle using a combination of polymers,lipids, and/or other biodegradable agents, such as, but not limited to,calcium phosphate. Components may be combined in a core-shell, hybrid,and/or layer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, primary construct andmmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller etal., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug DelivRev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Suet al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, primaryconstructs and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the polynucleotide, primaryconstruct and mmRNA of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol Ther. 2012 20:609-615). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to deliver polynucleotides, primary constructs andmmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., JContr Rel. 2006 111:368-370).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the polynucleotides, primary constructs and mmRNA of the presentinvention. The PEG-charge-conversional polymer may improve upon thePEG-polyanion block copolymers by being cleaved into a polycation atacidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer contain PEG may be used to delivery ofthe polynucleotide, primary construct and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferase-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031).

Peptides and Proteins

The polynucleotide, primary construct, and mmRNA of the invention can beformulated with peptides and/or proteins in order to increasetransfection of cells by the polynucleotide, primary construct, ormmRNA. In one embodiment, peptides such as, but not limited to, cellpenetrating peptides and proteins and peptides that enable intracellulardelivery may be used to deliver pharmaceutical formulations. Anon-limiting example of a cell penetrating peptide which may be usedwith the pharmaceutical formulations of the present invention includes acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci.62(16):1839-49 (2005), all of which are incorporated herein byreference). The compositions can also be formulated to include a cellpenetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. polynucleotides, primaryconstructs, and mmRNA of the invention may be complexed to peptidesand/or proteins such as, but not limited to, peptides and/or proteinsfrom Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics(Cambridge, Mass.) in order to enable intracellular delivery (Cronicanet al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 200973:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct, or mmRNA may beintroduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotide, primary construct, ormmRNA, alter the biodistribution of the polynucleotide, primaryconstruct, or mmRNA (e.g., by targeting specific tissues or cell types),and/or increase the translation of encoded protein.

Cells

The polynucleotide, primary construct, and mmRNA of the invention can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions mayinclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified RNA in virus-like particles (VLPs),and electroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than mmRNA have beendocumented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fanget al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., Proc NatlAcad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

Cell-based formulations of the polynucleotide, primary construct, andmmRNA of the invention may be used to ensure cell transfection (e.g., inthe cellular carrier), alter the biodistribution of the polynucleotide,primary construct, or mmRNA (e.g., by targeting the cell carrier tospecific tissues or cell types), and/or increase the translation ofencoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety).

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotide,primary construct, or mmRNA of the invention can include hyaluronidase,which catalyzes the hydrolysis of hyaluronan. By catalyzing thehydrolysis of hyaluronan, a constituent of the interstitial barrier,hyaluronidase lowers the viscosity of hyaluronan, thereby increasingtissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440;herein incorporated by reference in its entirety). It is useful to speedtheir dispersion and systemic distribution of encoded proteins producedby transfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a polynucleotide, primaryconstruct, or mmRNA of the invention administered intramuscularly orsubcutaneously.

Nanoparticle Mimics

The polynucleotide, primary construct or mmRNA of the invention may beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotide, primary construct or mmRNA of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 hereinincorporated by reference in its entirety).

Nanotubes

The polynucleotides, primary constructs or mmRNA of the invention can beattached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Thepolynucleotides, primary constructs or mmRNA may be bound to thenanotubes through forces such as, but not limited to, steric, ionic,covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,primary constructs or mmRNA into cells. The size and/or the surfacestructure of at least one nanotube may be altered so as to govern theinteraction of the nanotubes within the body and/or to attach or bind tothe polynucleotides, primary constructs or mmRNA disclosed herein. Inone embodiment, the building block and/or the functional groups attachedto the building block of the at least one nanotube may be altered toadjust the dimensions and/or properties of the nanotube. As anon-limiting example, the length of the nanotubes may be altered tohinder the nanotubes from passing through the holes in the walls ofnormal blood vessels but still small enough to pass through the largerholes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the polynucleotides, primary constructs or mmRNA may bemixed with pharmaceutically acceptable excipients and/or deliveryvehicles.

In one embodiment, the polynucleotides, primary constructs or mmRNA areattached and/or otherwise bound to at least one rosette nanotube. Therosette nanotubes may be formed by a process known in the art and/or bythe process described in International Publication No. WO2012094304,herein incorporated by reference in its entirety. At least onepolynucleotide, primary construct and/or mmRNA may be attached and/orotherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one polynucleotide, primary construct and/or mmRNA underconditions which may cause at least one polynucleotide, primaryconstruct or mmRNA to attach or otherwise bind to the rosette nanotubes.

Conjugates

The polynucleotides, primary constructs, and mmRNA of the inventioninclude conjugates, such as a polynucleotide, primary construct, ormmRNA covalently linked to a carrier or targeting group, or includingtwo encoding regions that together produce a fusion protein (e.g.,bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entirety.

In one embodiment, the conjugate of the present invention may functionas a carrier for the mmRNA of the present invention. The conjugate maycomprise a cationic polymer such as, but not limited to, polyamine,polylysine, polyalkylenimine, and polyethylenimine which may be graftedto with poly(ethylene glycol). As a non-limiting example, the conjugatemay be similar to the polymeric conjugate and the method of synthesizingthe polymeric conjugate described in U.S. Pat. No. 6,586,524 hereinincorporated by reference in its entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. Patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides,primary constructs or mmRNA with phosphorothioate backbones andoligonucleosides with other modified backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P(O)₂—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the polynucleotides featured herein havemorpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O—, S-, or N-alkyl; O—, S-, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotides,primary constructs or mmRNA include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties, or a group for improving the pharmacodynamicproperties, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the polynucleotide, primary construct, ormmRNA is covalently conjugated to a cell penetrating polypeptide. Thecell-penetrating peptide may also include a signal peptide sequence. Theconjugates of the invention can be designed to have increased stability;increased cell transfection; and/or altered the biodistribution (e.g.,targeted to specific tissues or cell types).

Self-Assembled Nucleic Acid Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393).

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM©), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of polynucleotides,primary constructs or mmRNA for any of therapeutic, pharmaceutical,diagnostic or imaging by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. Delivery may be nakedor formulated.

Naked Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be delivered to a cell naked. As used herein in, “naked”refers to delivering polynucleotides, primary constructs or mmRNA freefrom agents which promote transfection. For example, thepolynucleotides, primary constructs or mmRNA delivered to the cell maycontain no modifications. The naked polynucleotides, primary constructsor mmRNA may be delivered to the cell using routes of administrationknown in the art and described herein.

Formulated Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be formulated, using the methods described herein. Theformulations may contain polynucleotides, primary constructs or mmRNAwhich may be modified and/or unmodified. The formulations may furtherinclude, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides, primary constructs or mmRNA maybe delivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The polynucleotides, primary constructs or mmRNA of the presentinvention may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited toenteral, gastroenteral, epidural, oral, transdermal, epidural(peridural), intracerebral (into the cerebrum), intracerebroventricular(into the cerebral ventricles), epicutaneous (application onto theskin), intradermal, (into the skin itself), subcutaneous (under theskin), nasal administration (through the nose), intravenous (into avein), intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the polynucleotides, primaryconstructs or mmRNA of the present invention are described below.

Parenteral and Injectable Administration

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the polynucleotides,primary constructs or mmRNA of the invention may be formulated foradministration topically. The skin may be an ideal target site fordelivery as it is readily accessible. Gene expression may be restrictednot only to the skin, potentially avoiding nonspecific toxicity, butalso to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, primary constructs or mmRNA tothe skin: (i) topical application (e.g. for local/regional treatmentand/or applications); (ii) intradermal injection (e.g. forlocal/regional treatment and/or applications); and (iii) systemicdelivery (e.g. for treatment of dermatologic diseases that affect bothcutaneous and extracutaneous regions). polynucleotides, primaryconstructs or mmRNA can be delivered to the skin by several differentapproaches known in the art. Most topical delivery approaches have beenshown to work for delivery of DNA, such as but not limited to, topicalapplication of non-cationic liposome-DNA complex, cationic liposome-DNAcomplex, particle-mediated (gene gun), puncture-mediated genetransfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, primaryconstructs or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA compositions to be delivered in more thanone injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; herein incorporated by reference intheir entireties. Methods of enhancing the permeability of tissue aredescribed in U.S. Publication Nos. 20040171980 and 20040236268 and U.S.Pat. No. 6,190,315; herein incorporated by reference in theirentireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; herein incorporatedby reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels, foams,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Penetration Enhancers

In one embodiment, the polynucleotides, primary construct and mmRNA ofpresent invention may use various penetration enhancers to deliver thepolynucleotides, primary construct and mmRNA to at least one areaassociated with one or more hyperproliferative diseases, disorders orconditions. Most drugs are present in solution in both ionized andnonionized forms. However, usually only lipid soluble or lipophilicdrugs readily cross cell membranes. It has been discovered that evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition to aidingthe diffusion of non-lipophilic drugs across cell membranes, penetrationenhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail. Combinations of penetration enhancer may also beencompassed by the scope of the present invention, for example, fattyacids/salts in combination with bile acids/salts. Other non-limitingexamples of combinations of penetration enhancers include thecombination of sodium salt of lauric acid, capric acid and UDCA.

Surfactants

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of the polynucleotides, primaryconstructs and mmRNA through the mucosa is enhanced. In addition to bilesalts and fatty acids, these penetration enhancers include, for example,sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Fatty Acids

Various fatty acids and their derivatives which act as penetrationenhancers include, but are not limited to, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁-C₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile Salts

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935).Various natural bile salts, and their synthetic derivatives, act aspenetration enhancers. Thus the term “bile salts” includes any of thenaturally occurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, but are notlimited to, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofpolynucleotides, primary construct and mmRNA through the mucosa isenhanced. With regards to their use as penetration enhancers in thepresent invention, chelating agents have the added advantage of alsoserving as DNase inhibitors, as most characterized DNA nucleases requirea divalent metal ion for catalysis and are thus inhibited by chelatingagents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agentsof the invention include but are not limited to disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-Chelating Non-Surfactants

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of polynucleotides, primary construct and mmRNAthrough the alimentary mucosa (Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33). This class ofpenetration enhancers include, but are not limited to, unsaturatedcyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of polynucleotides, primary construct andmmRNA at the cellular level may also be added to the pharmaceutical andother compositions of the present invention. For example, cationiclipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188),cationic glycerol derivatives, and polycationic molecules, such aspolylysine (Lollo et al., PCT Application WO 97/30731), are also knownto enhance the cellular uptake of polynucleotides, primary construct andmmRNA.

Other agents may be utilized to enhance the penetration of theadministered polynucleotides, primary construct and mmRNA, includingglycols such as ethylene glycol and propylene glycol, pyrrols such as2-pyrrol, azones, and terpenes such as limonene and menthone.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the polynucleotides, primaryconstructs or mmRNA are spatially retained within or proximal to atarget tissue. Provided are method of providing a composition to atarget tissue of a mammalian subject by contacting the target tissue(which contains one or more target cells) with the composition underconditions such that the composition, in particular the nucleic acidcomponent(s) of the composition, is substantially retained in the targettissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissue. Advantageously, retentionis determined by measuring the amount of the nucleic acid present in thecomposition that enters one or more target cells. For example, at least1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.99% of the nucleic acids administered to thesubject are present intracellularly at a period of time followingadministration. For example, intramuscular injection to a mammaliansubject is performed using an aqueous composition containing aribonucleic acid and a transfection reagent, and retention of thecomposition is determined by measuring the amount of the ribonucleicacid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of apolynucleotides, primary constructs or mmRNA such that the polypeptideof interest is produced in at least one target cell. The compositionsgenerally contain a cell penetration agent, although “naked” nucleicacid (such as nucleic acids without a cell penetration agent or otheragent) is also contemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains polynucleotides, primary constructs or mmRNA characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentpolynucleotides, primary constructs or mmRNA, where one or more than oneof the polynucleotides, primary constructs or mmRNA encodes apolypeptide of interest. Optionally, the composition also contains acell penetration agent to assist in the intracellular delivery of thecomposition. A determination is made of the dose of the compositionrequired to produce the polypeptide of interest in a substantialpercentage of cells contained within the predetermined volume of thetarget tissue (generally, without inducing significant production of thepolypeptide of interest in tissue adjacent to the predetermined volume,or distally to the target tissue). Subsequent to this determination, thedetermined dose is introduced directly into the tissue of the mammaliansubject.

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA to be delivered in more than one injectionor by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir or patch pump. Non-limitingexamples of patch pumps include those manufactured and/or sold by BD®(Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMedTherapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.),UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeafTherapeutics (Boston, Mass.).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 m to 500 m. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention.

Payload Administration: Detectable Agents and Therapeutic Agents

The polynucleotides, primary constructs or mmRNA described herein can beused in a number of different scenarios in which delivery of a substance(the “payload”) to a biological target is desired, for example deliveryof detectable substances for detection of the target, or delivery of atherapeutic agent. Detection methods can include, but are not limitedto, both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides, primary constructs or mmRNA can be designed toinclude both a linker and a payload in any useful orientation. Forexample, a linker having two ends is used to attach one end to thepayload and the other end to the nucleobase, such as at the C-7 or C-8positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5positions of cytosine or uracil. The polynucleotide of the invention caninclude more than one payload (e.g., a label and a transcriptioninhibitor), as well as a cleavable linker. In one embodiment, themodified nucleotide is a modified 7-deaza-adenosine triphosphate, whereone end of a cleavable linker is attached to the C7 position of7-deaza-adenine, the other end of the linker is attached to an inhibitor(e.g., to the C5 position of the nucleobase on a cytidine), and a label(e.g., Cy5) is attached to the center of the linker (see, e.g., compound1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat.No. 7,994,304, incorporated herein by reference). Upon incorporation ofthe modified 7-deaza-adenosine triphosphate to an encoding region, theresulting polynucleotide having a cleavable linker attached to a labeland an inhibitor (e.g., a polymerase inhibitor). Upon cleavage of thelinker (e.g., with reductive conditions to reduce a linker having acleavable disulfide moiety), the label and inhibitor are released.Additional linkers and payloads (e.g., therapeutic agents, detectablelabels, and cell penetrating payloads) are described herein.

For example, the polynucleotides, primary constructs or mmRNA describedherein can be used in reprogramming induced pluripotent stem cells (iPScells), which can directly track cells that are transfected compared tototal cells in the cluster. In another example, a drug that may beattached to the polynucleotides, primary constructs or mmRNA via alinker and may be fluorescently labeled can be used to track the drug invivo, e.g. intracellularly. Other examples include, but are not limitedto, the use of polynucleotides, primary constructs or mmRNA inreversible drug delivery into cells.

The polynucleotides, primary constructs or mmRNA described herein can beused in intracellular targeting of a payload, e.g., detectable ortherapeutic agent, to specific organelle. Exemplary intracellulartargets can include, but are not limited to, the nuclear localizationfor advanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the polynucleotides, primary constructs or mmRNA describedherein can be used to deliver therapeutic agents to cells or tissues,e.g., in living animals. For example, the polynucleotides, primaryconstructs or mmRNA described herein can be used to deliver highly polarchemotherapeutics agents to kill cancer cells. The polynucleotides,primary constructs or mmRNA attached to the therapeutic agent through alinker can facilitate member permeation allowing the therapeutic agentto travel into a cell to reach an intracellular target.

In another example, the polynucleotides, primary constructs or mmRNA canbe attached to the polynucleotides, primary constructs or mmRNA a viralinhibitory peptide (VIP) through a cleavable linker. The cleavablelinker can release the VIP and dye into the cell. In another example,the polynucleotides, primary constructs or mmRNA can be attached throughthe linker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectableprecursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Combinations

The polynucleotides, primary constructs or mmRNA may be used incombination with one or more other therapeutic, prophylactic,diagnostic, or imaging agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the present disclosure. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of pharmaceutical, prophylactic, diagnostic, orimaging compositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. As anon-limiting example, the nucleic acids or mmRNA may be used incombination with a pharmaceutical agent for the treatment of cancer orto control hyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the nucleic acids andmmRNA of the present invention that encodes anti-proliferative moleculesmay be in a pharmaceutical composition with a lipopolymer (see e.g.,U.S. Pub. No. 20110218231, herein incorporated by reference in itsentirety, claiming a pharmaceutical composition comprising a DNA plasmidencoding an anti-proliferative molecule and a lipopolymer) which may beadministered with at least one chemotherapeutic or anticancer agent.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g., two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administeredin one dose/at one time/single route/single point of contact, i.e.,single administration event. As used herein, a “total daily dose” is anamount given or prescribed in 24 hr period. It may be administered as asingle unit dose. In one embodiment, the mmRNA of the present inventionare administered to a subject in split doses. The mmRNA may beformulated in buffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. Fatty acids such as oleicacid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotide,primary construct or mmRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administeredpolynucleotide, primary construct or mmRNA may be accomplished bydissolving or suspending the polynucleotide, primary construct or mmRNAin an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the polynucleotide, primary construct ormmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of polynucleotide, primary construct or mmRNAto polymer and the nature of the particular polymer employed, the rateof polynucleotide, primary construct or mmRNA release can be controlled.Examples of other biodegradable polymers include, but are not limitedto, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the polynucleotide, primaryconstruct or mmRNA in liposomes or microemulsions which are compatiblewith body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be use for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 m to 500 m. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof polynucleotides, primary constructs or mmRNA administered to amammal. Bioavailability can be assessed by measuring the area under thecurve (AUC) or the maximum serum or plasma concentration (C_(max)) ofthe unchanged form of a compound following administration of thecompound to a mammal. AUC is a determination of the area under the curveplotting the serum or plasma concentration of a compound along theordinate (Y-axis) against time along the abscissa (X-axis). Generally,the AUC for a particular compound can be calculated using methods knownto those of ordinary skill in the art and as described in G. S. Banker,Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,Marcel Dekker, New York, Inc., 1996, herein incorporated by reference.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotide, primary construct or mmRNA, measured as AUC,C_(max), or C_(min) in a mammal is greater, when co-administered with adelivery agent as described herein, than when such co-administrationdoes not take place. In some embodiments, the bioavailability of thepolynucleotide, primary construct or mmRNA can increase by at leastabout 2%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

Therapeutic Window

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in the therapeutic window of the administered polynucleotide,primary construct or mmRNA composition as compared to the therapeuticwindow of the administered polynucleotide, primary construct or mmRNAcomposition lacking a delivery agent as described herein. As used herein“therapeutic window” refers to the range of plasma concentrations, orthe range of levels of therapeutically active substance at the site ofaction, with a high probability of eliciting a therapeutic effect. Insome embodiments, the therapeutic window of the polynucleotide, primaryconstruct or mmRNA when co-administered with a delivery agent asdescribed herein can increase by at least about 2%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%.

Volume of Distribution

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit animproved volume of distribution (V_(dist)), e.g., reduced or targeted,relative to a composition lacking a delivery agent as described herein.The volume of distribution (V_(dist)) relates the amount of the drug inthe body to the concentration of the drug in the blood or plasma. Asused herein, the term “volume of distribution” refers to the fluidvolume that would be required to contain the total amount of the drug inthe body at the same concentration as in the blood or plasma: V_(dist)equals the amount of drug in the body/concentration of drug in blood orplasma. For example, for a 10 mg dose and a plasma concentration of 10mg/L, the volume of distribution would be 1 liter. The volume ofdistribution reflects the extent to which the drug is present in theextravascular tissue. A large volume of distribution reflects thetendency of a compound to bind to the tissue components compared withplasma protein binding. In a clinical setting, V_(dist) can be used todetermine a loading dose to achieve a steady state concentration. Insome embodiments, the volume of distribution of the polynucleotide,primary construct or mmRNA when co-administered with a delivery agent asdescribed herein can decrease at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50μg/ml may be preferred. For example, a protein expression of 50-200μg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290). Unlikeuntargeted mass spectrometry frequently used in biomarker discoverystudies, targeted MS methods are peptide sequence-based modes of MS thatfocus the full analytical capacity of the instrument on tens to hundredsof selected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12). The ionicspecies contained in solution may be transferred into the gas phase bydispersing a fine spray of charge droplets, evaporating the solvent andejecting the ions from the charged droplets to generate a mist of highlycharged droplets. The mist of highly charged droplets may be analyzedusing at least 1, at least 2, at least 3 or at least 4 mass analyzerssuch as, but not limited to, a quadropole mass analyzer. Further, themass spectrometry method may include a purification step. As anon-limiting example, the first quadrapole may be set to select a singlem/z ratio so it may filter out other molecular ions having a differentm/z ratio which may eliminate complicated and time-consuming samplepurification procedures prior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894). As non-limitingexamples, mass analyzers used in the MALDI analysis may include a lineartime-of-flight (TOF), a TOF reflectron or a Fourier transform massanalyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. Uses of Polynucleotides, Primary Constructs and mmRNA of theInvention

The polynucleotides, primary constructs and mmRNA of the presentinvention are designed, in preferred embodiments, to provide foravoidance or evasion of deleterious bio-responses such as the immuneresponse and/or degradation pathways, overcoming the threshold ofexpression and/or improving protein production capacity, improvedexpression rates or translation efficiency, improved drug or proteinhalf-life and/or protein concentrations, optimized protein localization,to improve one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, secretion efficiency (whenapplicable), accessibility to circulation, and/or modulation of a cell'sstatus, function and/or activity.

Therapeutics

Therapeutic Agents

The polynucleotides, primary constructs or mmRNA of the presentinvention, such as modified nucleic acids and modified RNAs, and theproteins translated from them described herein can be used astherapeutic or prophylactic agents. They are provided for use inmedicine. For example, polynucleotide, primary construct or mmRNAdescribed herein can be administered to a subject, wherein thepolynucleotide, primary construct or mmRNA is translated in vivo toproduce a therapeutic or prophylactic polypeptide in the subject.Provided are compositions, methods, kits, and reagents for diagnosis,treatment or prevention of a disease or condition in humans and othermammals. The active therapeutic agents of the invention includepolynucleotides, primary constructs or mmRNA, cells containingpolynucleotides, primary constructs or mmRNA or polypeptides translatedfrom the polynucleotides, primary constructs or mmRNA.

In certain embodiments, provided herein are combination therapeuticscontaining one or more polynucleotide, primary construct or mmRNAcontaining translatable regions that encode for a protein or proteinsthat boost a mammalian subject's immunity along with a protein thatinduces antibody-dependent cellular toxicity.

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the polynucleotide, primaryconstruct or mmRNA described herein. Such translation can be in vivo, exvivo, in culture, or in vitro. The cell population is contacted with aneffective amount of a composition containing nucleic acid that has atleast one nucleoside modification, and a translatable region encodingthe recombinant polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered polynucleotide, primaryconstruct or mmRNA directs production of one or more recombinantpolypeptides that provide a functional activity which is substantiallyabsent in the cell, tissue or organism in which the recombinantpolypeptide is translated. For example, the missing functional activitymay be enzymatic, structural, or gene regulatory in nature. In relatedembodiments, the administered polynucleotide, primary construct or mmRNAdirects production of one or more recombinant polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantpolypeptide is translated.

In other embodiments, the administered polynucleotide, primary constructor mmRNA directs production of one or more recombinant polypeptides thatreplace a polypeptide (or multiple polypeptides) that is substantiallyabsent in the cell in which the recombinant polypeptide is translated.Such absence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject; for example, due to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

In some embodiments, modified mRNAs and their encoded polypeptides inaccordance with the present invention may be used for treatment of anyof a variety of diseases, disorders, and/or conditions described herein.

VI. Kits and Devices

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides, primary constructs or mmRNA) of theinvention. In one embodiment, the kit comprises one or more functionalantibodies or function fragments thereof.

Said kits can be for protein production, comprising a firstpolynucleotide, primary construct or mmRNA comprising a translatableregion. The kit may further comprise packaging and instructions and/or adelivery agent to form a formulation composition. The delivery agent maycomprise a saline, a buffered solution, a lipidoid or any delivery agentdisclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium. In a further embodiment, the buffersolutions may be precipitated or it may be lyophilized. The amount ofeach component may be varied to enable consistent, reproducible higherconcentration saline or simple buffer formulations. The components mayalso be varied in order to increase the stability of modified RNA in thebuffer solution over a period of time and/or under a variety ofconditions. In one aspect, the present invention provides kits forprotein production, comprising: polynucleotide, primary construct ormmRNA comprising a translatable region, provided in an amount effectiveto produce a desired amount of a protein encoded by the translatableregion when introduced into a target cell; a second polynucleotidecomprising an inhibitory nucleic acid, provided in an amount effectiveto substantially inhibit the innate immune response of the cell; andpackaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the present invention provides kits for proteinproduction, comprising polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and a mammalian cellsuitable for translation of the translatable region of the first nucleicacid.

Devices

The present invention provides for devices which may incorporatepolynucleotides, primary constructs or mmRNA that encode polypeptides ofinterest. These devices contain in a stable formulation the reagents tosynthesize a polynucleotide in a formulation available to be immediatelydelivered to a subject in need thereof, such as a human patient.

In some embodiments the device is self-contained, and is optionallycapable of wireless remote access to obtain instructions for synthesisand/or analysis of the generated polynucleotide, primary construct ormmRNA. The device is capable of mobile synthesis of at least onepolynucleotide, primary construct or mmRNA and preferably an unlimitednumber of different polynucleotides, primary constructs or mmRNA. Incertain embodiments, the device is capable of being transported by oneor a small number of individuals. In other embodiments, the device isscaled to fit on a benchtop or desk. In other embodiments, the device isscaled to fit into a suitcase, backpack or similarly sized object. Inanother embodiment, the device may be a point of care or handhelddevice. In further embodiments, the device is scaled to fit into avehicle, such as a car, truck or ambulance, or a military vehicle suchas a tank or personnel carrier. The information necessary to generate amodified mRNA encoding polypeptide of interest is present within acomputer readable medium present in the device.

In one embodiment, a device may be used to assess levels of a proteinwhich has been administered in the form of polynucleotide, primaryconstruct or mmRNA. The device may comprise a blood, urine or otherbiofluidic test.

In some embodiments, the device is capable of communication (e.g.,wireless communication) with a database of nucleic acid and polypeptidesequences which may be nucleic acid and polypeptide sequences. Thedevice contains at least one sample block for insertion of one or moresample vessels. Such sample vessels are capable of accepting in liquidor other form any number of materials such as template DNA, nucleotides,enzymes, buffers, and other reagents. The sample vessels are alsocapable of being heated and cooled by contact with the sample block. Thesample block is generally in communication with a device base with oneor more electronic control units for the at least one sample block. Thesample block preferably contains a heating module, such heating moleculecapable of heating and/or cooling the sample vessels and contentsthereof to temperatures between about −20 C and above +100 C. The devicebase is in communication with a voltage supply such as a battery orexternal voltage supply. The device also contains means for storing anddistributing the materials for RNA synthesis.

Optionally, the sample block contains a module for separating thesynthesized nucleic acids. Alternatively, the device contains aseparation module operably linked to the sample block. Preferably thedevice contains a means for analysis of the synthesized nucleic acid.Such analysis includes sequence identity (demonstrated such as byhybridization), absence of non-desired sequences, measurement ofintegrity of synthesized mRNA (such has by microfluidic viscometrycombined with spectrophotometry), and concentration and/or potency ofmodified RNA (such as by spectrophotometry).

In certain embodiments, the device is combined with a means fordetection of pathogens present in a biological material obtained from asubject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) formicrobial identification.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

In some embodiments, the device may be a pump or comprise a catheter foradministration of compounds or compositions of the invention across theblood brain barrier. Such devices include but are not limited to apressurized olfactory delivery device, iontophoresis devices,multi-layered microfluidic devices, and the like. Such devices may beportable or stationary. They may be implantable or externally tetheredto the body or combinations thereof.

Devices for administration may be employed to deliver thepolynucleotides, primary constructs or mmRNA of the present inventionaccording to single, multi- or split-dosing regimens taught herein. Suchdevices are described below.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein.

A method for delivering therapeutic agents to a solid tissue has beendescribed by Bahrami et al. and is taught for example in US PatentPublication 20110230839, the contents of which are incorporated hereinby reference in their entirety. According to Bahrami, an array ofneedles is incorporated into a device which delivers a substantiallyequal amount of fluid at any location in said solid tissue along eachneedle's length.

A device for delivery of biological material across the biologicaltissue has been described by Kodgule et al. and is taught for example inUS Patent Publication 20110172610, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple hollow micro-needles made of one or more metals andhaving outer diameters from about 200 microns to about 350 microns andlengths of at least 100 microns are incorporated into the device whichdelivers peptides, proteins, carbohydrates, nucleic acid molecules,lipids and other pharmaceutically active ingredients or combinationsthereof.

A delivery probe for delivering a therapeutic agent to a tissue has beendescribed by Gunday et al. and is taught for example in US PatentPublication 20110270184, the contents of which are incorporated hereinby reference in their entirety. According to Gunday, multiple needlesare incorporated into the device which moves the attached capsulesbetween an activated position and an inactivated position to force theagent out of the capsules through the needles.

A multiple-injection medical apparatus has been described by Assaf andis taught for example in US Patent Publication 20110218497, the contentsof which are incorporated herein by reference in their entirety.According to Assaf, multiple needles are incorporated into the devicewhich has a chamber connected to one or more of said needles and a meansfor continuously refilling the chamber with the medical fluid after eachinjection.

In one embodiment, the polynucleotide, primary construct, or mmRNA isadministered subcutaneously or intramuscularly via at least 3 needles tothree different, optionally adjacent, sites simultaneously, or within a60 minutes period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10 sitessimultaneously or within a 60 minute period). The split doses can beadministered simultaneously to adjacent tissue using the devicesdescribed in U.S. Patent Publication Nos. 20110230839 and 20110218497,each of which is incorporated herein by reference.

An at least partially implantable system for injecting a substance intoa patient's body, in particular a penis erection stimulation system hasbeen described by Forsell and is taught for example in US PatentPublication 20110196198, the contents of which are incorporated hereinby reference in their entirety. According to Forsell, multiple needlesare incorporated into the device which is implanted along with one ormore housings adjacent the patient's left and right corpora cavernosa. Areservoir and a pump are also implanted to supply drugs through theneedles.

A method for the transdermal delivery of a therapeutic effective amountof iron has been described by Berenson and is taught for example in USPatent Publication 20100130910, the contents of which are incorporatedherein by reference in their entirety. According to Berenson, multipleneedles may be used to create multiple micro channels in stratum corneumto enhance transdermal delivery of the ionic iron on an iontophoreticpatch.

A method for delivery of biological material across the biologicaltissue has been described by Kodgule et al and is taught for example inUS Patent Publication 20110196308, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple biodegradable microneedles containing a therapeuticactive ingredient are incorporated in a device which delivers proteins,carbohydrates, nucleic acid molecules, lipids and other pharmaceuticallyactive ingredients or combinations thereof.

A transdermal patch comprising a botulinum toxin composition has beendescribed by Donovan and is taught for example in US Patent Publication20080220020, the contents of which are incorporated herein by referencein their entirety. According to Donovan, multiple needles areincorporated into the patch which delivers botulinum toxin under stratumcorneum through said needles which project through the stratum corneumof the skin without rupturing a blood vessel.

A small, disposable drug reservoir, or patch pump, which can holdapproximately 0.2 to 15 mL of liquid formulations can be placed on theskin and deliver the formulation continuously subcutaneously using asmall bore needed (e.g., 26 to 34 gauge). As non-limiting examples, thepatch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to34 gauge needle (BD™ Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mmby 17 mm with a 2 mL reservoir used for drug delivery such as insulin(OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMedTherapeutics, San Francisco, Calif.). Further, the patch pump may bebattery powered and/or rechargeable.

A cryoprobe for administration of an active agent to a location ofcryogenic treatment has been described by Toubia and is taught forexample in US Patent Publication 20080140061, the contents of which areincorporated herein by reference in their entirety. According to Toubia,multiple needles are incorporated into the probe which receives theactive agent into a chamber and administers the agent to the tissue.

A method for treating or preventing inflammation or promoting healthyjoints has been described by Stock et al and is taught for example in USPatent Publication 20090155186, the contents of which are incorporatedherein by reference in their entirety. According to Stock, multipleneedles are incorporated in a device which administers compositionscontaining signal transduction modulator compounds.

A multi-site injection system has been described by Kimmell et al. andis taught for example in US Patent Publication 20100256594, the contentsof which are incorporated herein by reference in their entirety.According to Kimmell, multiple needles are incorporated into a devicewhich delivers a medication into a stratum corneum through the needles.

A method for delivering interferons to the intradermal compartment hasbeen described by Dekker et al. and is taught for example in US PatentPublication 20050181033, the contents of which are incorporated hereinby reference in their entirety. According to Dekker, multiple needleshaving an outlet with an exposed height between 0 and 1 mm areincorporated into a device which improves pharmacokinetics andbioavailability by delivering the substance at a depth between 0.3 mmand 2 mm.

A method for delivering genes, enzymes and biological agents to tissuecells has described by Desai and is taught for example in US PatentPublication 20030073908, the contents of which are incorporated hereinby reference in their entirety. According to Desai, multiple needles areincorporated into a device which is inserted into a body and delivers amedication fluid through said needles.

A method for treating cardiac arrhythmias with fibroblast cells has beendescribed by Lee et al and is taught for example in US PatentPublication 20040005295, the contents of which are incorporated hereinby reference in their entirety. According to Lee, multiple needles areincorporated into the device which delivers fibroblast cells into thelocal region of the tissue.

A method using a magnetically controlled pump for treating a brain tumorhas been described by Shachar et al. and is taught for example in U.S.Pat. No. 7,799,012 (method) and 7799016 (device), the contents of whichare incorporated herein by reference in their entirety. AccordingShachar, multiple needles were incorporated into the pump which pushes amedicating agent through the needles at a controlled rate.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al. and are taught for examplein U.S. Pat. No. 8,029,496, the contents of which are incorporatedherein by reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A micro-needle transdermal transport device has been described by Angelet al and is taught for example in U.S. Pat. No. 7,364,568, the contentsof which are incorporated herein by reference in their entirety.According to Angel, multiple needles are incorporated into the devicewhich transports a substance into a body surface through the needleswhich are inserted into the surface from different directions. Themicro-needle transdermal transport device may be a solid micro-needlesystem or a hollow micro-needle system. As a non-limiting example, thesolid micro-needle system may have up to a 0.5 mg capacity, with300-1500 solid micro-needles per cm² about 150-700 μm tall coated with adrug. The micro-needles penetrate the stratum corneum and remain in theskin for short duration (e.g., 20 seconds to 15 minutes). In anotherexample, the hollow micro-needle system has up to a 3 mL capacity todeliver liquid formulations using 15-20 microneedles per cm2 beingapproximately 950 μm tall. The micro-needles penetrate the skin to allowthe liquid formulations to flow from the device into the skin. Thehollow micro-needle system may be worn from 1 to 30 minutes depending onthe formulation volume and viscosity.

A device for subcutaneous infusion has been described by Dalton et aland is taught for example in U.S. Pat. No. 7,150,726, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Dalton, multiple needles are incorporated into the device whichdelivers fluid through the needles into a subcutaneous tissue.

A device and a method for intradermal delivery of vaccines and genetherapeutic agents through microcannula have been described by Miksztaet al. and are taught for example in U.S. Pat. No. 7,473,247, thecontents of which are incorporated herein by reference in theirentirety. According to Mitszta, at least one hollow micro-needle isincorporated into the device which delivers the vaccines to thesubject's skin to a depth of between 0.025 mm and 2 mm.

A method of delivering insulin has been described by Pettis et al and istaught for example in U.S. Pat. No. 7,722,595, the contents of which areincorporated herein by reference in their entirety. According to Pettis,two needles are incorporated into a device wherein both needles insertessentially simultaneously into the skin with the first at a depth ofless than 2.5 mm to deliver insulin to intradermal compartment and thesecond at a depth of greater than 2.5 mm and less than 5.0 mm to deliverinsulin to subcutaneous compartment.

Cutaneous injection delivery under suction has been described byKochamba et al. and is taught for example in U.S. Pat. No. 6,896,666,the contents of which are incorporated herein by reference in theirentirety. According to Kochamba, multiple needles in relative adjacencywith each other are incorporated into a device which injects a fluidbelow the cutaneous layer.

A device for withdrawing or delivering a substance through the skin hasbeen described by Down et al and is taught for example in U.S. Pat. No.6,607,513, the contents of which are incorporated herein by reference intheir entirety. According to Down, multiple skin penetrating memberswhich are incorporated into the device have lengths of about 100 micronsto about 2000 microns and are about 30 to 50 gauge.

A device for delivering a substance to the skin has been described byPalmer et al and is taught for example in U.S. Pat. No. 6,537,242, thecontents of which are incorporated herein by reference in theirentirety. According to Palmer, an array of micro-needles is incorporatedinto the device which uses a stretching assembly to enhance the contactof the needles with the skin and provides a more uniform delivery of thesubstance.

A perfusion device for localized drug delivery has been described byZamoyski and is taught for example in U.S. Pat. No. 6,468,247, thecontents of which are incorporated herein by reference in theirentirety. According to Zamoyski, multiple hypodermic needles areincorporated into the device which injects the contents of thehypodermics into a tissue as said hypodermics are being retracted.

A method for enhanced transport of drugs and biological molecules acrosstissue by improving the interaction between micro-needles and human skinhas been described by Prausnitz et al. and is taught for example in U.S.Pat. No. 6,743,211, the contents of which are incorporated herein byreference in their entirety. According to Prausnitz, multiplemicro-needles are incorporated into a device which is able to present amore rigid and less deformable surface to which the micro-needles areapplied.

A device for intraorgan administration of medicinal agents has beendescribed by Ting et al and is taught for example in U.S. Pat. No.6,077,251, the contents of which are incorporated herein by reference intheir entirety. According to Ting, multiple needles having side openingsfor enhanced administration are incorporated into a device which byextending and retracting said needles from and into the needle chamberforces a medicinal agent from a reservoir into said needles and injectssaid medicinal agent into a target organ.

A multiple needle holder and a subcutaneous multiple channel infusionport has been described by Brown and is taught for example in U.S. Pat.No. 4,695,273, the contents of which are incorporated herein byreference in their entirety. According to Brown, multiple needles on theneedle holder are inserted through the septum of the infusion port andcommunicate with isolated chambers in said infusion port.

A dual hypodermic syringe has been described by Horn and is taught forexample in U.S. Pat. No. 3,552,394, the contents of which areincorporated herein by reference in their entirety. According to Horn,two needles incorporated into the device are spaced apart less than 68mm and may be of different styles and lengths, thus enabling injectionsto be made to different depths.

A syringe with multiple needles and multiple fluid compartments has beendescribed by Hershberg and is taught for example in U.S. Pat. No.3,572,336, the contents of which are incorporated herein by reference intheir entirety. According to Hershberg, multiple needles areincorporated into the syringe which has multiple fluid compartments andis capable of simultaneously administering incompatible drugs which arenot able to be mixed for one injection.

A surgical instrument for intradermal injection of fluids has beendescribed by Eliscu et al. and is taught for example in U.S. Pat. No.2,588,623, the contents of which are incorporated herein by reference intheir entirety. According to Eliscu, multiple needles are incorporatedinto the instrument which injects fluids intradermally with a widerdisperse.

An apparatus for simultaneous delivery of a substance to multiple breastmilk ducts has been described by Hung and is taught for example in EP1818017, the contents of which are incorporated herein by reference intheir entirety. According to Hung, multiple lumens are incorporated intothe device which inserts though the orifices of the ductal networks anddelivers a fluid to the ductal networks.

A catheter for introduction of medications to the tissue of a heart orother organs has been described by Tkebuchava and is taught for examplein WO2006138109, the contents of which are incorporated herein byreference in their entirety. According to Tkebuchava, two curved needlesare incorporated which enter the organ wall in a flattened trajectory.

Devices for delivering medical agents have been described by Mckay etal. and are taught for example in WO2006118804, the content of which areincorporated herein by reference in their entirety. According to Mckay,multiple needles with multiple orifices on each needle are incorporatedinto the devices to facilitate regional delivery to a tissue, such asthe interior disc space of a spinal disc.

A method for directly delivering an immunomodulatory substance into anintradermal space within a mammalian skin has been described by Pettisand is taught for example in WO2004020014, the contents of which areincorporated herein by reference in their entirety. According to Pettis,multiple needles are incorporated into a device which delivers thesubstance through the needles to a depth between 0.3 mm and 2 mm.

Methods and devices for administration of substances into at least twocompartments in skin for systemic absorption and improvedpharmacokinetics have been described by Pettis et al. and are taught forexample in WO2003094995, the contents of which are incorporated hereinby reference in their entirety. According to Pettis, multiple needleshaving lengths between about 300 μm and about 5 mm are incorporated intoa device which delivers to intradermal and subcutaneous tissuecompartments simultaneously.

A drug delivery device with needles and a roller has been described byZimmerman et al. and is taught for example in WO2012006259, the contentsof which are incorporated herein by reference in their entirety.According to Zimmerman, multiple hollow needles positioned in a rollerare incorporated into the device which delivers the content in areservoir through the needles as the roller rotates.

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the mmRNA of the present invention on a single, multi- orsplit dosing schedule. Such methods and devices are described below.

A catheter-based delivery of skeletal myoblasts to the myocardium ofdamaged hearts has been described by Jacoby et al and is taught forexample in US Patent Publication 20060263338, the contents of which areincorporated herein by reference in their entirety. According to Jacoby,multiple needles are incorporated into the device at least part of whichis inserted into a blood vessel and delivers the cell compositionthrough the needles into the localized region of the subject's heart.

An apparatus for treating asthma using neurotoxin has been described byDeem et al and is taught for example in US Patent Publication20060225742, the contents of which are incorporated herein by referencein their entirety. According to Deem, multiple needles are incorporatedinto the device which delivers neurotoxin through the needles into thebronchial tissue.

A method for administering multiple-component therapies has beendescribed by Nayak and is taught for example in U.S. Pat. No. 7,699,803,the contents of which are incorporated herein by reference in theirentirety. According to Nayak, multiple injection cannulas may beincorporated into a device wherein depth slots may be included forcontrolling the depth at which the therapeutic substance is deliveredwithin the tissue.

A surgical device for ablating a channel and delivering at least onetherapeutic agent into a desired region of the tissue has been describedby McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096,the contents of which are incorporated herein by reference in theirentirety. According to McIntyre, multiple needles are incorporated intothe device which dispenses a therapeutic agent into a region of tissuesurrounding the channel and is particularly well suited fortransmyocardial revascularization operations.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al and are taught for example inU.S. Pat. No. 8,029,496, the contents of which are incorporated hereinby reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A device and a method for delivering fluid into a flexible biologicalbarrier have been described by Yeshurun et al. and are taught forexample in U.S. Pat. No. 7,998,119 (device) and U.S. Pat. No. 8,007,466(method), the contents of which are incorporated herein by reference intheir entirety. According to Yeshurun, the micro-needles on the devicepenetrate and extend into the flexible biological barrier and fluid isinjected through the bore of the hollow micro-needles.

A method for epicardially injecting a substance into an area of tissueof a heart having an epicardial surface and disposed within a torso hasbeen described by Bonner et al and is taught for example in U.S. Pat.No. 7,628,780, the contents of which are incorporated herein byreference in their entirety. According to Bonner, the devices haveelongate shafts and distal injection heads for driving needles intotissue and injecting medical agents into the tissue through the needles.

A device for sealing a puncture has been described by Nielsen et al andis taught for example in U.S. Pat. No. 7,972,358, the contents of whichare incorporated herein by reference in their entirety. According toNielsen, multiple needles are incorporated into the device whichdelivers a closure agent into the tissue surrounding the puncture tract.

A method for myogenesis and angiogenesis has been described by Chiu etal. and is taught for example in U.S. Pat. No. 6,551,338, the contentsof which are incorporated herein by reference in their entirety.According to Chiu, 5 to 15 needles having a maximum diameter of at least1.25 mm and a length effective to provide a puncture depth of 6 to 20 mmare incorporated into a device which inserts into proximity with amyocardium and supplies an exogeneous angiogenic or myogenic factor tosaid myocardium through the conduits which are in at least some of saidneedles.

A method for the treatment of prostate tissue has been described byBolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, thecontents of which are incorporated herein by reference in theirentirety. According to Bolmsj, a device comprising a catheter which isinserted through the urethra has at least one hollow tip extendible intothe surrounding prostate tissue. An astringent and analgesic medicine isadministered through said tip into said prostate tissue.

A method for infusing fluids to an intraosseous site has been describedby Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726,the contents of which are incorporated herein by reference in theirentirety. According to Findlay, multiple needles are incorporated into adevice which is capable of penetrating a hard shell of material coveredby a layer of soft material and delivers a fluid at a predetermineddistance below said hard shell of material.

A device for injecting medications into a vessel wall has been describedby Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863,the contents of which are incorporated herein by reference in theirentirety. According to Vigil, multiple injectors are mounted on each ofthe flexible tubes in the device which introduces a medication fluidthrough a multi-lumen catheter, into said flexible tubes and out of saidinjectors for infusion into the vessel wall.

A catheter for delivering therapeutic and/or diagnostic agents to thetissue surrounding a bodily passageway has been described by Faxon etal. and is taught for example in U.S. Pat. No. 5,464,395, the contentsof which are incorporated herein by reference in their entirety.According to Faxon, at least one needle cannula is incorporated into thecatheter which delivers the desired agents to the tissue through saidneedles which project outboard of the catheter.

Balloon catheters for delivering therapeutic agents have been describedby Orr and are taught for example in WO2010024871, the contents of whichare incorporated herein by reference in their entirety. According toOrr, multiple needles are incorporated into the devices which deliverthe therapeutic agents to different depths within the tissue.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the mmRNA of the present invention according to the single,multi- or split dosing regimens taught herein. Such methods and devicesare described below.

An electro collagen induction therapy device has been described byMarquez and is taught for example in US Patent Publication 20090137945,the contents of which are incorporated herein by reference in theirentirety. According to Marquez, multiple needles are incorporated intothe device which repeatedly pierce the skin and draw in the skin aportion of the substance which is applied to the skin first.

An electrokinetic system has been described by Etheredge et al. and istaught for example in US Patent Publication 20070185432, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Etheredge, micro-needles are incorporated into a device which drivesby an electrical current the medication through the needles into thetargeted treatment site.

An iontophoresis device has been described by Matsumura et al. and istaught for example in U.S. Pat. No. 7,437,189, the contents of which areincorporated herein by reference in their entirety. According toMatsumura, multiple needles are incorporated into the device which iscapable of delivering ionizable drug into a living body at higher speedor with higher efficiency.

Intradermal delivery of biologically active agents by needle-freeinjection and electroporation has been described by Hoffmann et al andis taught for example in U.S. Pat. No. 7,171,264, the contents of whichare incorporated herein by reference in their entirety. According toHoffmann, one or more needle-free injectors are incorporated into anelectroporation device and the combination of needle-free injection andelectroporation is sufficient to introduce the agent into cells in skin,muscle or mucosa.

A method for electropermeabilization-mediated intracellular delivery hasbeen described by Lundkvist et al. and is taught for example in U.S.Pat. No. 6,625,486, the contents of which are incorporated herein byreference in their entirety. According to Lundkvist, a pair of needleelectrodes is incorporated into a catheter. Said catheter is positionedinto a body lumen followed by extending said needle electrodes topenetrate into the tissue surrounding said lumen. Then the deviceintroduces an agent through at least one of said needle electrodes andapplies electric field by said pair of needle electrodes to allow saidagent pass through the cell membranes into the cells at the treatmentsite.

A delivery system for transdermal immunization has been described byLevin et al. and is taught for example in WO2006003659, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Levin, multiple electrodes are incorporated into the device whichapplies electrical energy between the electrodes to generate microchannels in the skin to facilitate transdermal delivery.

A method for delivering RF energy into skin has been described bySchomacker and is taught for example in WO2011163264, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Schomacker, multiple needles are incorporated into a device whichapplies vacuum to draw skin into contact with a plate so that needlesinsert into skin through the holes on the plate and deliver RF energy.

VII. Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C6 alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments,polynucleotide, primary construct or mmRNA of the present invention maybe considered biologically active if even a portion of thepolynucleotide, primary construct or mmRNA is biologically active ormimics an activity considered biologically relevant.

Cancer: As used herein, the term “cancer” in a subject refers to thepresence of cells possessing characteristics, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Often, cancer cells will be in the form of a tumor, but such cells mayexist alone within a subject, or may circulate in the blood stream asindependent cells, such as leukemic cells.

Cell growth: As used herein, the term “cell growth” is principallyassociated with growth in cell numbers, which occurs by means of cellreproduction (i.e. proliferation) when the rate of the latter is greaterthan the rate of cell death (e.g. by apoptosis or necrosis).

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NH^(R1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons. In some embodiment, thecycloalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl);(3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C3-s cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁-6thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C3-s cycloalkyl; (10) C₁₋₆ alk-C3-scycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C3-s cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Condition: As used herein, the term “condition” refers to a disorderthat presents with observable symptoms.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery ofpolynucleotide, primary construct or mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Disease: As used herein, the term “disease” refers to an abnormalcondition affecting the body of an organism often showing specificbodily symptoms.

Disorder: As used herein, the term “disorder,” refers to a disruption ofor an interference with normal functions or established systems of thebody.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dose splittingfactor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Encodedprotein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide, primary construct or mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Genotype: As used herein, “genotype” refers to the change in thegenotype, or genetic makeup, of a subject, cell, tissue, organ and/ororganism.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more polynucleotides, primary constructs, or mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondinclude an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

Metastasis: As used herein, the term “metastasis” means the process bywhich cancer spreads from the place at which it first arose as a primarytumor to distant locations in the body.

Method of Treating: The phrase “a method of treating” or its equivalent,when applied to, for example, cancer refers to a procedure or course ofaction that is designed to reduce or eliminate the number of cancercells, prevent the increase in the number of cancer cells, or toalleviate the symptoms of a cancer in a subject. A method of treatingcancer or another disorder does not necessarily mean that the cancercells or other disorder will, in fact, be completely eliminated, thatthe number of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of a subject, is nevertheless deemed anoverall beneficial course of action.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutical composition: The phrase “pharmaceutical composition”refers to a composition that alters the etiology of a disease, disorderand/or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Phenotype: As used herein, “phenotype” refers to the set of observablecharacteristics of a subject, cell, tissue, organ and/or organism.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Progression: As used herein, the term “progression” (e.g., cancerprogression) means the advancement or worsening of or toward a diseaseor condition.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Regression: As used herein, the term “regression” or “degree ofregression” refers to the reversal, either phenotypically orgenotypically, of a cancer progression. Slowing or stopping cancerprogression may be considered regression.

Reducing the effect: As used herein, the phrase “reducing the effect”when referring to symptoms, means reducing, eliminating or alleviatingthe symptom in the subject. It does not necessarily mean that thesymptom will, in fact, be completely eliminated, reduced or alleviated.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Side effect: As used herein, the phrase “side effect” refers to asecondary effect of treatment.

Signal Peptide Sequences: As used herein, the phrase “signal peptidesequences” refers to a sequence which can direct the transport orlocalization of a protein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Skin: The term “skin” is the thin layer of tissue forming the naturalouter covering of the body of a subject and includes the epidermis andthe dermis. The dermis is the thick layer of living tissue below theepidermis which is the surface epithelium of the skin.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Symptom: As used herein, the term “symptom” is a signal of a disease,disorder and/or condition. For example, symptoms may be felt or noticedby the subject who has them but may not be easily accessed by looking ata subject's outward appearance or behaviors. Examples of symptomsinclude, but are not limited to, weakness, aches and pains, fever,fatigue, weight loss, blood clots, increased blood calcium levels, lowwhite blood cell count, short of breath, dizziness, headaches,hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth,change in bowel habits, change in bladder function, long-lasting sores,white patches inside the mouth, white spots on the tongue, unusualbleeding or discharge, thickening or lump on parts of the body,indigestion, trouble swallowing, changes in warts or moles, change innew skin and nagging cough or hoarseness.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Tumor: As used herein, a “tumor” is an abnormal growth of tissue,whether benign or malignant.

Tumor growth: As used herein, the term “tumor growth” or “tumormetastases” means an increased mass or volume of the tumor or expansionof the tumor distribution.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Polynucleotide Production

Modified mrnas (mmRNA) according to the invention may be made usingstandard laboratory methods and materials.

The open reading frame with various upstream or downstream regions(3-globin, tags, etc.) is ordered from DNA2.0 (Menlo Park, Calif.) andtypically contains a multiple cloning site with XbaI recognition. Uponreceipt of the construct, it is reconstituted and transformed intochemically competent E. coli. For the present invention, NEB DH5-alphaCompetent E. coli are used. A typical clone map is shown in FIG. 7 .Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

-   -   1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3. Place the mixture on ice for 30 minutes. Do not mix.    -   4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5. Place on ice for 5 minutes. Do not mix.    -   6. Pipette 950 μl of room temperature SOC into the mixture.    -   7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8. Warm selection plates to 37° C.    -   9. Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmidis first linearized using a restriction enzyme such as XbaI. A typicalrestriction digest with XbaI will comprise the following: Plasmid 1.0μg; 10× Buffer 1.0 μl; XbaI 1.5 μl; dH₂O up to 10 μl; incubated at 37°C. for 1 hr. If performing at lab scale (<5 μg), the reaction is cleanedup using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) permanufacturer's instructions. Larger scale purifications may need to bedone with a product that has a larger load capacity such as Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). Following the cleanup,the linearized vector is quantified using the NanoDrop and analyzed toconfirm linearization using agarose gel electrophoresis.

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 4. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 4.

TABLE 4 G-CSF Sequences SEQ ID NO Description  9 cDNAsequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 10cDNA having T7 polymerase site, AfeI and Xba restriction site:TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA11 Optimized sequence; containing T7 polymerase site, AfeI and Xbarestriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA12 mRNA sequence (transcribed)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC CAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC GUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGA AG

Example 2: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 uM) 0.75 μl; ReversePrimer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O diluted to 25.0μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3. In Vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1. Template cDNA 1.0 μg 2. 10x transcription buffer (400 mM Tris-HCl pH8.0, 2.0 μl 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3. Custom NTPs(25 mM each) 7.2 μl 4. RNase Inhibitor 20 U 5. T7 RNA polymerase 3000 U6. dH₂0 Up to 20.0 μl. and 7. Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4. Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5. PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g., about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6. Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCAcap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7. Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 9; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 12with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA (3′ O-Me-m7G(5′)ppp(5′)G) cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 9; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 12with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 9; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 12with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 9; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 12with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8. Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9. Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 10. Formulation of Polynucleotides

Polynucleotides may be formulated for in vitro and in vivo experimentsaccording to the methods taught in International Application No.PCT/US12/069610 filed Dec. 14, 2012, the contents of which areincorporated herein by reference in their entirety.

Example 11. Assays and Methods of Detection or Analysis ofPolynucleotides

Polynucleotides may be investigated using the methods described inInternational Application No. PCT/US2012/58519 filed Oct. 3, 2012 and inInternational Application No. PCT/US12/069610 filed Dec. 14, 2012, thecontents of which are incorporated herein by reference in theirentirety.

Example 12. Carboxy-Terminal Peptide Constructs

The polynucleotide, primary construct and/or mmRNA of the presentinvention can have at least one nucleic acid sequence encoding at leastone carboxy-terminal peptide (CTP). The carboxy-terminal peptides canincrease the half-life of the polynucleotides, primary constructs and/ormmRNA of the present invention and can increase the half-life of theproteins encoded by the polynucleotides, primary constructs and/ormmRNA. The polynucleotides, primary constructs and/or mmRNA of thepresent invention may include at least one chemical modification and/orat least one terminal modification (e.g., miR binding sites). Examplesof carboxy-terminal peptide constructs are shown in Table 5. In Table 5,the carboxy-terminal peptides are underlined in the amino acidsequences, “CTP” stands for carboxy-terminal peptide, “FGF23” stands forfibroblast growth factor 23, “HGH” stands for human growth hormone,“G-CSF” stands for granulocyte colony stimulating growth factor and“GLP” stands for glucagon-like peptide.

TABLE 5 Carboxy-terminal peptide constructs Protein SEQ ID SEQ IDDescription Sequence NO mRNA Sequence NO Albiglutide MKWVSFISLLF 13(without LFSSAYSGSLD albumin) KRHGEGTFTSD with 2 CTPs VSSYLEGQAAKEFIAWLVKGRS SSSKAPPPSLPS PSRLPGPSDTPI LPQSSSSKAPPP SLPSPSRLPGPS DTPILPQAlbiglutide GGGAAAUAAGAGAGAAAAGAAG 30 (without AGUAAGAAGAAAUAUAAGAGCCalbumin) ACCAUGAAGUGGGUGUCGUUCA with 2 CTPs UCUCACUGUUGUUCCUUUUCAGCUCGGCGUACUCGGGGUCACUCG ACAAACGCCACGGAGAGGGCACC UUUACUUCCGAUGUCAGCUCCUACCUCGAAGGCCAGGCGGCAAA GGAAUUCAUCGCCUGGCUGGUG AAGGGAAGAAGCUCAUCGAGCAAGGCCCCUCCACCGUCCCUCCCU UCGCCGUCCCGGCUGCCGGGACC AAGCGACACUCCGAUCCUGCCACAAUCGUCAUCCUCCAAAGCUCCU CCACCCUCGCUGCCAUCCCCGUC AAGGCUGCCCGGUCCGAGCGAUACCCCGAUUCUCCCGCAGUCAUC CUCGAGCAAGGCCCCUCCGCCCU CACUGCCAUCGCCAAGCCGCCUGCCGGGACCUUCCGACACCCCGAU CCUCCCGCAGUCAUCCUCGAGCA AGGCCCCUCCGCCCUCACUGCCAUCGCCAAGCCGCCUGCCGGGACC UUCCGACACCCCGAUCCUCCCGC AGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC Albiglutide MKWVSFISLLF 14 GLP-1 withLFSSAYSGSLD 2 CTPs KRHGEGTFTSD VSSYLEGQAAK EFIAWLVKGRS SSSKAPPPSLPSPSRLPGPSDTPI LPQSSSSKAPPP SLPSPSRLPGPS DTPILPQ Dulaglutide MKWVSFISLLF15 GLP-1 with LFSSAYSGSLD 2 CTPs KRHGEGTFTSD VSSYLEEQAAK EFIAWLVKGGGGGGGSGGGGS GGGGSSSSKAP PPSLPSPSRLPG PSDTPILPQSSS SKAPPPSLPSPSRLPGPSDTPILP Q FGF23 MLGARLRLWV 16 GGGAAAUAAGAGAGAAAAGAAG 31 fragmentCALCSVCSMSV AGUAAGAAGAAAUAUAAGAGCC (180-251) LRASAEDDSERACCAUGCUGGGUGCAAGACUUC with 1 CTP DPLNVLKPRAR GCCUUUGGGUGUGCGCACUUUGMTPAPASCSQE CAGCGUGUGUUCAAUGAGCGUG LPSAEDNSPMA CUGAGAGCAUCCGCCGAGGACGSDPLGVVRGGR ACUCCGAACGUGACCCCUUGAAC VNTHAGGTGP GUGCUGAAGCCUAGGGCUAGAAEGCRPFAKFISS UGACGCCAGCCCCUGCGUCAUGC SSKAPPPSLPSPUCGCAAGAACUCCCGUCGGCGGA SRLPGPSDTPIL GGAUAACUCCCCUAUGGCAUCCG MAUCCUCUGGGAGUGGUGCGAGG UGGUCGUGUUAACACCCACGCG GGUGGCACUGGACCCGAAGGGUGUAGACCUUUCGCAAAGUUUAU CUCAUCCUCGAGCAAGGCCCCUC CGCCCUCACUGCCAUCGCCAAGCCGCCUGCCGGGACCUUCCGACAC CCCGAUCCUCCCGCAGGCUGGAG CCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACC CCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC FGF23 MKWVSFISLLF 17 fragment LFSSAYSGSLD (180-251)KRSAEDDSERD with 2 CTPs PLNVLKPRAR MTPAPASCSQE LPSAEDNSPMA SDPLGVVRGGRVNTHAGGTGP EGCRPFAKFISS SSKAPPPSLPSP SRLPGPSDTPIL PQSSSSKAPPPSLPSPSRLPGPSD TPILPQ FGF23 MLGARLRLWV 18 fragment CALCSVCSMSV (180-251)LRASAEDDSER with 1 CTP DPLNVLKPRAR MTPAPASCSQE LPSAEDNSPMA SDPLGVVRGGRVNTHAGGTGP EGCRPFAKFISS SSKAPPPSLPSP SRLPGPSDTPIL M G-CSF withMAGPATQSPM 19 GGGAAAUAAGAGAGAAAAGAAG 32 3 CTPs KLMALQLLLWAGUAAGAAGAAAUAUAAGAGCC HSALWTVQESS ACCAUGGCCGGUCCCGCGACCCA SSKAPPPSLPSPAAGCCCCAUGAAACUUAUGGCCC SRLPGPSDTPIL UGCAGUUGCUGCUUUGGCACUC PQATPLGPASSGGCCCUCUGGACAGUCCAAGAA LPQSFLLKCLE UCAUCCUCGAGCAAGGCCCCUCC QVRKIQGDGAGCCCUCACUGCCAUCGCCAAGCC ALQEKLCATY GCCUGCCGGGACCUUCCGACACC KLCHPEELVLLCCGAUCCUCCCGCAGGCGACUCC GHSLGIPWAPL UCUCGGACCUGCCUCAUCGUUGC SSCPSQALQLACGCAGUCAUUCCUUUUGAAGUG GCLSQLHSGLF UCUGGAGCAGGUGCGAAAGAUU LYQGLLQALEGCAGGGCGAUGGAGCCGCACUCCA ISPELGPTLDTL AGAGAAGCUCUGCGCGACAUAC QLDVADFATTIAAACUUUGCCAUCCCGAGGAGC WQQMEELGM UCGUACUGCUCGGGCACAGCUU APALQPTQGAGGGGAUUCCCUGGGCUCCUCUCU MPAFASAFQRR CGUCCUGUCCGUCGCAGGCUUUG AGGVLVASHLCAGUUGGCAGGGUGCCUUUCCC QSFLEVSYRVL AGCUCCACUCCGGUUUGUUCUU RHLAQPSSSSKGUAUCAGGGACUGCUGCAAGCC APPPSLPSPSRL CUUGAGGGAAUCUCGCCAGAAU PGPSDTPILPQSUGGGCCCGACGCUGGACACGUU SSSKAPPPSLPS GCAGCUCGACGUGGCGGAUUUC PSRLPGPSDTPIGCAACAACCAUCUGGCAGCAGA LPQ UGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGG CAAUGCCGGCCUUUGCGUCCGCG UUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAA UCAUUUUUGGAAGUCUCGUACC GGGUGCUGAGACAUCUUGCGCAGCCGUCAUCCUCGAGCAAGGCCC CUCCGCCCUCACUGCCAUCGCCA AGCCGCCUGCCGGGACCUUCCGACACCCCGAUCCUCCCGCAGUCAU CCUCGAGCAAGGCCCCUCCGCCC UCACUGCCAUCGCCAAGCCGCCUGCCGGGACCUUCCGACACCCCGA UCCUCCCGCAGGCUGGAGCCUCG GUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCC CCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGC G-CSF with MATGSRTSLLL 20 GGGAAAUAAGAGAGAAAAGAAG 33 3 CTPsAFGLLCLPWLQ AGUAAGAAGAAAUAUAAGAGCC EGSASSSSKAPP ACCAUGGCCACUGGGUCGAGGAPSLPSPSRLPGP CCAGCCUGUUGUUGGCCUUUGG SDTPILPQATPL GCUGCUUUGUCUGCCAUGGCUCCGPASSLPQSFLL AAGAGGGAUCCGCAUCAUCCUC KCLEQVRKTQG GAGCAAGGCCCCUCCGCCCUCACDGAALQEKLC UGCCAUCGCCAAGCCGCCUGCCG ATYKLCHPEEL GGACCUUCCGACACCCCGAUCCUVLLGHSLGIPW CCCGCAGGCGACUCCUCUCGGAC APLSSCPSQAL CUGCCUCAUCGUUGCCGCAGUCAQLAGCLSQLHS UUCCUUUUGAAGUGUCUGGAGC GLFLYQGLLQA AGGUGCGAAAGAUUCAGGGCGALEGISPELGPTL UGGAGCCGCACUCCAAGAGAAG DTLQLDVADF CUCUGCGCGACAUACAAACUUUATTIWQQMEEL GCCAUCCCGAGGAGCUCGUACUG GIVIAPALQPTQ CUCGGGCACAGCUUGGGGAUUCGAMPAFASAF CCUGGGCUCCUCUCUCGUCCUGU QRRAGGVLVA CCGUCGCAGGCUUUGCAGUUGGSHLQSFLEVSY CAGGGUGCCUUUCCCAGCUCCAC RVLRHLAQPSS UCCGGUUUGUUCUUGUAUCAGGSSKAPPPSLPSP GACUGCUGCAAGCCCUUGAGGG SRLPGPSDTPIL AAUCUCGCCAGAAUUGGGCCCGPQSSSSKAPPPS ACGCUGGACACGUUGCAGCUCG LPSPSRLPGPSD ACGUGGCGGAUUUCGCAACAACTPILPQ CAUCUGGCAGCAGAUGGAGGAA CUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGG CCUUUGCGUCCGCGUUUCAGCGC AGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUU GGAAGUCUCGUACCGGGUGCUG AGACAUCUUGCGCAGCCGUCAUCCUCGAGCAAGGCCCCUCCGCCCU CACUGCCAUCGCCAAGCCGCCUG CCGGGACCUUCCGACACCCCGAUCCUCCCGCAGUCAUCCUCGAGCA AGGCCCCUCCGCCCUCACUGCCA UCGCCAAGCCGCCUGCCGGGACCUUCCGACACCCCGAUCCUCCCGC AGGCUGGAGCCUCGGUGGCCAU GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUG AAUAAAGUCUGAGUGGGCGGCG-CSF with MAGPATQSPM 21 GGGAAAUAAGAGAGAAAAGAAG 34 1 CTP KLMALQLLLWAGUAAGAAGAAAUAUAAGAGCC HSALWTVQESS ACCAUGGCCGGUCCCGCGACCCA SSKAPPPSLPSPAAGCCCCAUGAAACUUAUGGCCC SRLPGPSDTPIL UGCAGUUGCUGCUUUGGCACUC PQMATPLGPASSGGCCCUCUGGACAGUCCAAGAA LPQSFLLKCLE UCAUCCUCGAGCAAGGCCCCUCC QVRKIQGDGAGCCCUCACUGCCAUCGCCAAGCC ALQEKLCATY GCCUGCCGGGACCUUCCGACACC KLCHPEELVLLCCGAUCCUCCCGCAGGCGACUCC GHSLGIPWAPL UCUCGGACCUGCCUCAUCGUUGC SSCPSQALQLACGCAGUCAUUCCUUUUGAAGUG GCLSQLHSGLF UCUGGAGCAGGUGCGAAAGAUU LYQGLLQALEGCAGGGCGAUGGAGCCGCACUCCA ISPELGPTLDTL AGAGAAGCUCUGCGCGACAUAC QLDVADFATTIAAACUUUGCCAUCCCGAGGAGC WQQMEELGM UCGUACUGCUCGGGCACAGCUU APALQPTQGAGGGGAUUCCCUGGGCUCCUCUCU MPAFASAFQRR CGUCCUGUCCGUCGCAGGCUUUG AGGVLVASHLCAGUUGGCAGGGUGCCUUUCCC QSFLEVSYRVL AGCUCCACUCCGGUUUGUUCUU RHLAQPGUAUCAGGGACUGCUGCAAGCC CUUGAGGGAAUCUCGCCAGAAU UGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUC GCAACAACCAUCUGGCAGCAGA UGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGG CAAUGCCGGCCUUUGCGUCCGCG UUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAA UCAUUUUUGGAAGUCUCGUACC GGGUGCUGAGACAUCUUGCGCAGCCGGCUGGAGCCUCGGUGGCCA UGCUUCUUGCCCCUUGGGCCUCC CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUU GAAUAAAGUCUGAGUGGGCGGC G-CSF with MAGPATQSPM 22GGGAAAUAAGAGAGAAAAGAAG 35 2 CTPs KLMALQLLLW AGUAAGAAGAAAUAUAAGAGCCHSALWTVQEA ACCAUGGCCGGUCCCGCGACCCA TPLGPASSLPQS AAGCCCCAUGAAACUUAUGGCCCFLLKCLEQVRK UGCAGUUGCUGCUUUGGCACUC IQGDGAALQEK GGCCCUCUGGACAGUCCAAGAALCATYKLCHPE GCGACUCCUCUCGGACCUGCCUC ELVLLGHSLGIP AUCGUUGCCGCAGUCAUUCCUUWAPLSSCPSQA UUGAAGUGUCUGGAGCAGGUGC LQLAGCLSQLH GAAAGAUUCAGGGCGAUGGAGCSGLFLYQGLLQ CGCACUCCAAGAGAAGCUCUGCG ALEGISPELGPT CGACAUACAAACUUUGCCAUCCCLDTLQLDVADF GAGGAGCUCGUACUGCUCGGGC ATTIWQQMEEL ACAGCUUGGGGAUUCCCUGGGCGIVIAPALQPTQ UCCUCUCUCGUCCUGUCCGUCGC GAMPAFASAF AGGCUUUGCAGUUGGCAGGGUGQRRAGGVLVA CCUUUCCCAGCUCCACUCCGGUU SHLQSFLEVSY UGUUCUUGUAUCAGGGACUGCURVLRHLAQPSS GCAAGCCCUUGAGGGAAUCUCG SSKAPPPSLPSP CCAGAAUUGGGCCCGACGCUGGSRLPGPSDTPIL ACACGUUGCAGCUCGACGUGGC PQSSSSKAPPPS GGAUUUCGCAACAACCAUCUGGLPSPSRLPGPSD CAGCAGAUGGAGGAACUGGGGA TPILPQ UGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUG CGUCCGCGUUUCAGCGCAGGGCG GGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGU CUCGUACCGGGUGCUGAGACAU CUUGCGCAGCCGUCAUCCUCGAGCAAGGCCCCUCCGCCCUCACUGC CAUCGCCAAGCCGCCUGCCGGGA CCUUCCGACACCCCGAUCCUCCCGCAGUCAUCCUCGAGCAAGGCCC CUCCGCCCUCACUGCCAUCGCCA AGCCGCCUGCCGGGACCUUCCGACACCCCGAUCCUCCCGCAGGCUG GAGCCUCGGUGGCCAUGCUUCU UGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG UACCCCCGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGCHGH with 3 MATGSRTSLLL 23 GGGAAAUAAGAGAGAAAAGAAG 36 CTPs AFGLLCLPWLQAGUAAGAAGAAAUAUAAGAGCC EGSASSSSKAPP ACCAUGGCCACUGGGUCGAGGA PSLPSPSRLPGPCCAGCCUGUUGUUGGCCUUUGG SDTPILPQFPTIP GCUGCUUUGUCUGCCAUGGCUCC LSRLFDNAMLRAAGAGGGAUCCGCAUCAUCCUC AHRLHQLAFDT GAGCAAGGCCCCUCCGCCCUCAC YQEFEEAYIPKUGCCAUCGCCAAGCCGCCUGCCG EQKYSFLQNPQ GGACCUUCCGACACCCCGAUCCUTSLCFSESIPTPS CCCGCAGUUCCCGACAAUUCCCC NREETQQKSNL UCUCAAGACUGUUUGAUAACGCELLRISLLLIQS UAUGCUCCGAGCCCACCGGCUGC WLEPVQFLRSV ACCAGCUGGCGUUCGAUACAUAFANSLVYGASD CCAAGAAUUCGAGGAAGCAUAC SNVYDLLKDLE AUCCCCAAAGAGCAGAAGUAUUEGIQTLMGRLE CGUUCCUUCAAAAUCCUCAGACA DGSPRTGQIFK UCGCUUUGUUUCUCGGAGUCAAQTYSKFDTNSH UUCCGACGCCCAGCAAUAGGGA NDDALLKNYG AGAAACGCAGCAGAAGUCGAACLLYCFRKDMD CUUGAGUUGUUGCGGAUUAGCU KVETFLRIVQC UGCUCCUGAUCCAGUCAUGGCUCRSVEGSCGFSS GAACCGGUGCAGUUCUUGCGCU SSKAPPPSLPSP CGGUGUUUGCGAACUCCCUGGUSRLPGPSDTPIL AUAUGGUGCGUCCGAUTJCAAAU PQSSSSKAPPPS GUCUACGACUUGCUCAAGGAUCLPSPSRLPGPSD UUGAAGAGGGGAUCCAAACUCU TPILPQ CAUGGGUAGGCUUGAGGACGGCUCGCCUCGCACGGGACAGAUCUU UAAGCAGACGUAUUCGAAAUUU GACACCAAUUCACAUAACGACGACGCGUUGCUCAAAAACUAUGG AUUGCUCUACUGCUUUCGGAAG GACAUGGAUAAAGUGGAGACAUUCUUGAGAAUCGUCCAGUGCAG AUCCGUAGAGGGAUCAUGCGGU UUUUCAUCCUCGAGCAAGGCCCCUCCGCCCUCACUGCCAUCGCCAA GCCGCCUGCCGGGACCUUCCGAC ACCCCGAUCCUCCCGCAGUCAUCCUCAGCAAGGCCCCUCCGCCCU CACUGCCAUCGCCAAGCCGCCUG CCGGGACCUUCCGACACCCCGAUCCUCCCGCAGGCUGGAGCCUCGG UGGCCAUGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUG GUCUUUGAAUAAAGUCUGAGUG GGCGGC HGH with 3MATGSRTSLLL 24 CTPs AFGLLCLPWLQ EGSASSSSKAPP PSLPFPTIPLSRL FDNAMLRAHRLHQLAFDTYQE FEEAYIPKEQK YSFLQNPQTSL CFSESIPTPSNR EETQQKSNLELLRISLLLIQSWL EPVQFLRSVFA NSLVYGASDSN VYDLLKDLEEG IQTLMGRLEDG SPRTGQIFKQTYSKFDTNSHND DALLKNYGLL YCFRKDMDKV ETFLRIVQCRS VEGSCGFSSSS KAPPPSLPSPSRLPGPSDTPILPQ SSSSKAPPPSLP SPSRLPGPSDTP ILPQ HUH with 3 MKWVSFISLLF 25CTPs LFSSAYSGSLD KRSSSSKAPPPS LPSPSRLPGPSD TPILPQFPTIPLS RLFDNAMLRAHRLHQLAFDTY QEFEEAYIPKE QKYSFLQNPQT SLCFSESIPTPS NREETQQKSNLELLRISLLLIQS WLEPVQFLRSV FANSLVYGASD SNVYDLLKDLE EGIQTLMGRLE DGSPRTGQIFKQTYSKFDTNSH NDDALLKNYG LLYCFRKDMD KVETFLRIVQC RSVEGSCGFSS SSKAPPPSLPSPSRLPGPSDTPIL PQSSSSKAPPPS LPSPSRLPGPSD TPILPQ GLP-1 with MKWVSFISLLF 262 CTP LFSSAYSGSLD KRHAEGTFTSD VSSYLEGQAAK EFIAWLVKGRS SSSKAPPPSLPSPSRLPGPSDTPI LPQSSSSKAPPP SLPSPSRLPGPS DTPILPQ GLP-2 MKWVSFISLLF 27Teduglutide LFSSAYSGSLD with 2 CTPs KRHGDGSFSDE MNTILDNLAAR DFINWLIQTKITDSSSSKAPPPSL PSPSRLPGPSDT PILPQSSSSKAP PPSLPSPSRLPG PSDTPILPQ 2 GLP-2MKWVSFISLLF 28 Teduglutide LFSSAYSGSLD with 2 CTPs KRHGDGSFSDEMNTILDNLAAR DFINWLIQTKIT DHGDGSFSDE MNTILDNLAAR DFINWLIQTKITDSSSSKAPPPSL PSPSRLPGPSDT PILPQSSSSKAP PPSLPSPSRLPG PSDTPILPQ GLP-2MATGSRTSLLL 29 Teduglutide AFGLLCLPWLQ with 3 CTPs EGSASSSSKAPPPSLPSPSRLPGP SDTPILPQHGD GSFSDEMNTIL DNLAARDFIN WLIQTKITDSSSSKAPPPSLPSPS RLPGPSDTPILP QSSSSKAPPPSL PSPSRLPGPSDT PILPQ

Example 13. Evaluation of Carboxy-Terminal Peptide Constructs In Vitro

mRNAs, such as those described in Table 5 which further comprise a polyAtail of at least 140 nucleotides and a 5′cap of Cap1, encoding a proteinof interest and at least one carboxy-terminal peptide are formulated insaline, a buffer, a lipid, a lipid nanoparticle, or lipofectamine 2000for analysis in vitro. The mRNA contains chemical modifications such as,but not limited to, fully modified with 5-methlcytosine andpseudouridine, fully modified with 5-methlycytosine and1-methylpseudouridine, fully modified with 1-methylpseudouridine, fullymodified with pseudouridine or where 25% of the uridine residues aremodified with 2-thiouridine and 25% of the cytosine residues aremodified with 5-methylcytosine. Varying concentrations (e.g., 0.5 mg/kg,0.05 mg/kg or 0.005 mg/kg) of the mRNA in the formulations aretransfected by methods described herein and known in the art.Measurements are taken at predetermined intervals (e.g., 2 hours, 8hours, 12 hours, 18 hours 24 hours and 48 hours) to determine the levelsof protein and/or mRNA in the sample.

Example 14. Evaluation of Carboxy-Terminal Peptide Constructs in Mammals

mRNAs, such as those described in Table 5 which further comprise a polyAtail of at least 140 nucleotides and a 5′cap of Cap1, encoding a proteinof interest and at least one carboxy-terminal peptide are formulated insaline, a buffer, a lipid, a lipid nanoparticle, or surgical sealantsfor delivery intramuscularly, intravenously and/or subcutaneously tomammals. The mRNA contains chemical modifications such as, but notlimited to, fully modified with 5-methlcytosine and pseudouridine, fullymodified with 5-methlycytosine and 1-methylpseudouridine, fully modifiedwith 1-methylpseudouridine, fully modified with pseudouridine or where25% of the uridine residues are modified with 2-thiouridine and 25% ofthe cytosine residues are modified with 5-methylcytosine. Varyingconcentrations (e.g., 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg) of the mRNAin the formulations are administered to the mammals by methods describedherein and/or known in the art. Measurements are taken at predeterminedintervals (e.g., 2 hours, 8 hours, 12 hours, 18 hours 24 hours and 48hours) to determine the levels of protein and/or mRNA in the sample.

Example 15. Evaluation of FGF23 In Vitro

mRNA encoding the full length of the wild type FGF23 sequence, mRNAencoding the C-terminal fragment (amino acid 180-251) of the wild-typeFGF23 with a native signal peptide, mRNA encoding the C-terminalfragment (amino acid 180-251) of the wild-type FGF23 with a non-nativesignal peptide (such as the HSA/KEX2 signal peptide), mRNA encoding theC-terminal fragment (amino acid 180-251) of the wild-type FGF23 with anative signal peptide and a C-terminal CTP and/or mRNA encoding theC-terminal fragment (amino acid 180-251) of the wild-type FGF23 with anative signal peptide and a N-terminal CTP is evaluated in renalproximal tubule epithelial cells by the methods described herein and/orare known in the art (see e.g., Medici et al. FGF-23-Klotho signalingstimulates proliferation and prevents vitamin D-induced apoptosis. J.Cell Biol. 2008: 182(3) 459-465; and Goetz et al. Isolated C-terminaltail of FGF23 alleviates hypophosphatemia by inhibitingFGF23-FGFR-Klotho complex formation. PNAS 2010: 107(1) 407-412; each ofwhich is herein incorporated by reference in its entirety). Thesupernatant from the transfections is assessed by ELISA for FGF23bioactivity for protein phosphorylation and cell proliferation.

Example 16. Evaluation of FGF23 In Vivo

mRNA encoding the full length of the wild type FGF23 sequence (aminoacid sequence shown in SEQ ID NO: 37), mRNA encoding the C-terminalfragment (amino acid 180-251) of the wild-type FGF23 with a nativesignal peptide, mRNA encoding the C-terminal fragment (amino acid180-251) of the wild-type FGF23 with a non-native signal peptide (suchas the HSA/KEX2 signal peptide), mRNA encoding the C-terminal fragment(amino acid 180-251) of the wild-type FGF23 with a native signal peptideand a C-terminal CTP and/or mRNA encoding the C-terminal fragment (aminoacid 180-251) of the wild-type FGF23 with a native signal peptide and aN-terminal CTP is formulated in a lipid nanoparticle. The formulatedmRNA is administered via a single intravenous injection to mammals at apredetermined dose (e.g., 0.005 mg/kg, 0.05 mg/kg or 0.5 mg/kg). Thelevels of FGF23 in the serum are measured by ELISA. The serum inorganicphosphate (Pi) is measured pre-dose and 1 hour, 3 hours and 6 hoursafter administration. The pharmacokinetics of each mRNA will beevaluated by methods known in the art and/or described herein.

Example 17. Human Growth Hormone Carboxy-Terminal Peptide Studies

Carboxy-Terminal Peptides (CTPs) were evaluated as a potential approachfor prolonging the half-life of therapeutic protein molecules. mRNAsencoding human growth hormone (hGH) were prepared using the in vitrotranscription method for these studies.

A. In Vitro Study

Human cervical carcinoma (HeLa) or hepatocellular carcinoma epithelial(HepG2) cells were seeded at density of 200,000 on 24-well plates incell culture medium with 10% fetal calf serum (FCS) overnight and nextday the growth media was replaced with a fresh one with no serum and nophenol red.

The cells were transfected with 384 ng of mRNA encoding the wild typesequence of hGH (mRNA sequence shown in SEQ ID NO: 38; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′Cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine) (wild typehGH) lipoplexed with 1.5 LF2000 per well or 500 ng of mRNA encoding thehGH sequence with one CTP (C-terminal peptide of the beta chain of humanChorionic Gonadotropin) at N terminus and two CTP (C-terminal peptide ofthe beta chain of human Chorionic Gonadotropin) at the C terminus (mRNAsequence shown in SEQ ID NO: 39; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (CTP modified hGH). Apositive control of mRNA encoding hGH (amino acid sequence shown in SEQID NO: 40; mRNA sequence shown in SEQ ID NO: 41; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′Cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine) was used andPBS was used as a negative control.

Two sets of aliquots culture media supernatants were collected 7 hourspost transfection. One set of samples were used to perform hGH ELISA todetermine the hGH protein level in the supernatants. As shown in Table6, the hGH ELISA detected high level of protein from the supernatantsfrom both the wild type hGH and the CTP modified hGH and also from thepositive control. The negative control (PBS) showed no signal.

TABLE 6 In Vitro hGH protein expression Protein Molecular ProteinExpression (ng/ul) Mass (kDa) HeLa Cells HepG2 Cells Wild type hGH 22967.3 318.5 CTP modified hGH 47.5 311.8 65.8 Positive control 22 662.6348.1 Negative control — 0 0

The second set of samples were used to perform a bioactivity test(Bioassay lab-Germany). The bioactivity assay was a cell basedproliferation assay using the NB2-11 cell line (rat lymphoma cells) todetermine the bioactivity of hGH. The cells were incubated 48 hours andcell proliferation was stimulated in a 96-well plate by linking hGH to aprolactin-receptor. There were 9 titration steps with one standardversus one sample on one plate. Proliferation dependent staining bywater soluble tetrazolium (WST) was conducted and bioactivity responsewas measured by the photometric absorbance of formazan signal (OD) inthe proliferation assay. The results are shown in Tables 7, 8 and 9.

TABLE 7 Bioactivity of HeLa cell vehicle vs. Standard (NIBSC WHO 98/574)Dose Vehicle from HeLa Cell Standard (NIBSC WHO 98/574) Response (OD)(OD) 1 287 2118.3 0.667 288.3 1933.3 0.444 293.7 1699.3 0.296 299 1422.70.198 295.7 1183.7 0.132 295 968 0.088 297 786 0.059 298.7 620.3 0.039300 573.3

TABLE 8 Bioactivity of Wild type hGH (HeLa) vs. Standard (NIBSC WHO98/574) Dose Wild type hGH (HeLa) Standard (NIBSC WHO 98/574) Response(OD) (OD) 1 2152.7 2108.3 0.667 2123 1939.7 0.444 1927 1647.3 0.2961697.7 1462 0.198 1463.7 1184.3 0.132 1212 959 0.088 968.7 777 0.059792.3 646 0.039 663 551

TABLE 9 Bioactivity of CTP modified hGH (HeLa) vs. Standard (NIBSC WHO98/574) Dose CTP modified hGH (HeLa) Standard (NIBSC WHO 98/574)Response (OD) (OD) 1 2107 2088.3 0.667 1970.3 1932.7 0.444 1804.7 1655.30.296 1572 1425.7 0.198 1286.7 1180.7 0.132 1048.3 966.3 0.088 858.3800.3 0.059 683.7 647.3 0.039 592.7 571.3

The results which summarized in Tables 7-9 above showed that mRNAsencoding wild type hGH and CTP modified hGH expressed proteins havepositive bioactivity response compare to the WHO hGH standard (NIBSC WHO98/574). The NB-2-II rat lymphoma cells showed strong proliferation 48hours after the addition of the supernatants from HeLa cells containingwild type hGH or CTP modified hGH proteins and no proliferation wasobserved from the vehicle supernatants.

B. In Vivo Study

Mice (n=4; C57BL/6; approximate weight: 0.025 kg/mouse) wereadministered intravenously mRNA encoding the wild type sequence of hGH(mRNA sequence shown in SEQ ID NO: 38; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (wild type hGH) formulatedin an LNP described in Table 10 (characterization in Table 11), mRNAencoding the hGH sequence with one CTP (C-terminal peptide of the betachain of human Chorionic Gonadotropin) at N terminus and two CTP(C-terminal peptide of the beta chain of human Chorionic Gonadotropin)at the C terminus (mRNA sequence shown in SEQ ID NO: 41; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′Cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine) (CTP modifiedhGH) formulated in an LNP described in Table 10 or a negative control ofPBS (vehicle only) as outlined in Table 12. The dose of CTP modified hGHwas corrected due to the size difference in mRNA of wild type hGH inorder to make the doses equimolar.

TABLE 10 LNP Formulation Characterization DLin-KC2-DMA DSPC CholesterolPEG-DMG Mole Percent 50.0 10.0 38.5 1.5 (mol %)

TABLE 11 LNP Formulation Characterization Encap- Drug Zeta at sulationConcen- Lipid:mRNA Mean size pH 7.4 (%) tration mRNA Ratio (nm) (mV)(Ribogreen) (mg/mL) Wild type 20:1 91.5 0.9 80 0.15 hGH PDI: 0.09 CTP20:1 82.6 0.9 81 0.14 modified PDI: 0.06 hGH

TABLE 12 Dosing Regimen Injection Cationic Dose volume Lipid Cohorts(mg/kg) (mL) Wild type KC2 7 0.05 0.1 hGH CTP KC2 7 0.065 0.1 modifiedhGH PBS none 2 none 0.1

The cohorts of mice administered the LNP formulated mRNA were bledterminally at 1 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hoursand 48 hours after administration and the PBS administered mice werebled terminally at 1 hour and 30 hours after administration to determinethe Human growth hormone (hGH) protein expression by ELISA. As shown inTable 13, hGH protein expression was detectable at least out to 24 hoursfor the mice that received a dose of 0.065 mg/kg of CTP modified hGHmice and at least out to 12 hours for the wild types hGH mice. Noprotein was detected in the negative PBS control. In Table 13, “NT”means not tested.

TABLE 13 Serum Protein Expression in Mice Wild type hGH CTP modified hGHPBS (pg/ml) (pg/ml) (pg/ml) 1 hour 0 0 0 6 hours 322.5 342 NT 12 hours109.4 133.6 NT 18 hours 0 77 NT 24 hours 0 0 NT 30 hours 0 0 0 48 hours0 0 NT

The mice serum hGH protein concentration (pg/ml) in the time coursecurve show that the mice administered the CTP modified hGH mRNAdemonstrated prolonged half-life as compared to the mice administeredwild type hGH mRNA.

Example 18. Human Granulocyte Colony-Stimulating Factor Carboxy-TerminalPeptide Study

Carboxy-Terminal Peptides (CTPs) were evaluated as a potential approachfor prolonging the half-life of therapeutic protein molecules. mRNAsencoding human granulocyte colony-stimulating factor (hGCSF) wereprepared using the in vitro transcription method for these studies.

Mice (n=4; C57BL/6; approximate weight: 0.025 kg/mouse) wereadministered intravenously mRNA encoding the wild type sequence of hGCSF(mRNA sequence shown in SEQ ID NO: 42; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (wild type hGCSF) formulatedin an LNP described in Table 14 (characterization in Table 15), mRNAencoding the hGCSF sequence with one CTP (C-terminal peptide of the betachain of human Chorionic Gonadotropin) at N terminus (mRNA sequenceshown in SEQ ID NO: 43; polyA tail of approximately 140 nucleotides notshown in sequence; 5′Cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) (1×CTP modified hGCSF) formulated in an LNPdescribed in Table 14 (characterization in Table 15), mRNA encoding thehGCSF sequence two CTP (C-terminal peptide of the beta chain of humanChorionic Gonadotropin) at the C terminus (C-terminal peptide of thebeta chain of human Chorionic Gonadotropin) (mRNA sequence shown in SEQID NO: 44; polyA tail of approximately 140 nucleotides not shown insequence; 5′Cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) (2×CTP modified hGCSF) formulated in an LNPdescribed in Table 14 (characterization in Table 15) or a negativecontrol of PBS as outlined in Table 16. The dose of 1×CTP modified hGCSFand 2×CTP modified hGCSF was corrected due to the size differences inmRNA in order to make the doses equimolar.

TABLE 14 LNP Formulation Characterization DLin-KC2-DMA DSPC CholesterolPEG-DMG Mole Percent 50.0 10.0 38.5 1.5 (mol %)

TABLE 15 LNP Formulation Characterization Zeta Lipid: Mean atEncapsulation Drug mRNA size pH 7.4 (%) Concentration mRNA Ratio (nm)(mV) (Ribogreen) (mg/mL) Wild type 20:1 82.3 0.7 77 0.14 hGCSF PDI: 0.041 × CTP 20:1 82.7 0.9 92 0.16 modified PDI: 0.06 hGCSF 2 × CTP 20:1 86.42.0 98 0.17 modified PDI: 0.07 hGCSF

TABLE 16 Dosing Regimen Injection Cationic Dose volume Lipid Cohorts(mg/kg) (mL) Wild type hGCSF KC2 7 0.05  0.1 1 × CTP modified hGCSF KC27 0.055 0.1 2 × CTP modified hGCSF KC2 7 0.060 0.1 PBS none 2 none 0.1

The cohorts of mice administered the LNP formulated mRNA were bledterminally at 1 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hoursand 48 hours after administration and the PBS administered mice werebled terminally at 1 hour and 30 hours after administration to determinethe human granulocyte colony-stimulating factor (hGCSF) proteinexpression in serum by ELISA. Blood was collected from the cohorts ofmice at 24 hours and 48 hours after administration to measure CBC(neutrophil level) as a pharmacological readout.

As shown in Table 17, hGCSF protein expression was detectable in serumat least out to 30 hours for the group treated with wild type hGCSFmRNA, 1×CTP modified hGCSF mRNA and 2×CTP modified hGCSF mRNA. Highoverall performance was seen with the 1×CTP modified hGCSF mRNA ascompared to the wild type hGCSF mRNA group and the 2×CTP modified hGCSFmRNA group. No protein was detected in the negative PBS control. InTable 17, “NT” means not tested.

TABLE 17 Serum Protein Expression in Mice Wild type 1 × CTP modified 2 ×CTP modified hGCSF hGCSF hGCSF PBS (pg/ml) (pg/ml) (pg/ml) (pg/ml) 1hour 0 0 0 0 6 hours 29743.2 56847.6 35343 NT 12 hours 29891.8 36333.234517.3 NT 18 hours 9046.7 22124.4 7375.8 NT 24 hours 12401.1 16149.18922.4 NT 30 hours 8936 5407.8 2646.8 0 48 hours 0 0 0 NT

Wild type hGCSF mRNA, 1×CTP modified hGCSF mRNA and 2×CTP modified hGCSFmRNA groups expressed a good amount of protein through the time points.The overall hGCSF protein concentration (pg/ml) in mice serum throughthe time course curve demonstrated a superior profile of 1×CTP modifiedhGCSF as compared to wild type hGCSF post a single IV administration ofLNP formulated mRNA.

As shown in Table 18, CBC (Neutrophil level) was measured as apharmacological readout. To demonstrate the specificity of the actionCBC (Neutrophil Level) blood was collected and evaluated from miceinjected with 0.065 mg/kg unrelated mRNA (mRNA encoding the hGH sequencewith one CTP (C-terminal peptide of the beta chain of human ChorionicGonadotropin) at N terminus and two CTP (C-terminal peptide of the betachain of human Chorionic Gonadotropin) at the C terminus (mRNA sequenceshown in SEQ ID NO: 39; polyA tail of approximately 140 nucleotides notshown in sequence; 5′Cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) (CTP modified hGH)) formulated in a LNP describedin Table 18 (characterization in Table 19).

TABLE 18 LNP Formulation Characterization DLin-KC2-DMA DSPC CholesterolPEG-DMG Mole Percent 50.0 10.0 38.5 1.5 (mol %)

TABLE 19 LNP Formulation Characterization Zeta Lipid: Mean atEncapsulation Drug mRNA size pH 7.4 (%) Concentration mRNA Ratio (nm)(mV) (Ribogreen) (mg/mL) CTP 20:1 82.6 0.9 81 0.14 modified PDI: 0.06hGH

Results of the neutrophil count are shown in Table 20 and neutrophilpercent are shown in Table 21.

TABLE 20 Neutrophil Count Neutrophil Count Neutrophil Count (K/ul)(K/ul) 24 Hours 48 Hours Wild type hGCSF 1.29 1.24 1 × CTP modifiedhGCSF 2.26 1.26 2 × CTP modified hGCSF 1.80 0.67 CTP modified hGH 0.720.33 PBS 0.35 0.47

TABLE 21 Absolute Neutrophil Percent Neutrophil Percent NeutrophilPercent (%) (%) 24 Hours 48 Hours Wild type hGCSF 25.5 15.2 1 × CTPmodified hGCSF 30.1 13.2 2 × CTP modified hGCSF 30.7 11.2 CTP modifiedhGH 12.1 9.7 PBS 10.4 12.8

Wild type hGCSF showed significant induction of neutrophil in mice at 24hours, around 3.7 fold compared to the vehicle (PBS). The 1×CTP modifiedhGCSF and 2×CTP modified hGCSF showed higher induction 6.5 and 5.1 foldrespectively at 24 hours as compared to the vehicle (PBS). Theneutrophil percent is also higher in the groups injected with the 1×CTPmodified hGCSF mRNA and 2×CTP modified hGCSF mRNA as compared to thewild type hGCSF and around three fold compared to the PBS. The inductionof the neutrophil counts and the neutrophil percent decreased at 48hours. The results from untreated group showed closely similar to thePBS.

Example 19. Human GLP-1 Carboxy-Terminal Peptide Study

Carboxy-Terminal Peptides (CTPs) were evaluated as a potential approachfor prolonging the half-life of therapeutic protein molecules. mRNAsencoding human GLP-1 were prepared using the in vitro transcriptionmethod for these studies.

Rats (n=6; Sprague Dawley rats with fitted jugular vein catheter(JVC-Harlan); approximate weight: 300-330 gm/rat) were administeredintravenously mRNA encoding the wild type sequence of human GLP1 (mRNAsequence shown in SEQ ID NO: 45; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (wild type hGLP1) formulatedin an LNP described in Table 22 (characterization in Table 23), mRNAencoding the human GLP1 sequence with one CTP (C-terminal peptide of thebeta chain of human Chorionic Gonadotropin) at C terminus (amino acidsequence shown in SEQ ID NO: 46; mRNA sequence shown in SEQ ID NO: 47;polyA tail of approximately 140 nucleotides not shown in sequence;5′Cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) (1×CTP modified hGLP1) formulated in an LNPdescribed in Table 22 (characterization in Table 23) or a negativecontrol of PBS (vehicle only) as outlined in Table 24. The dose of 1×CTPmodified hGLP1 was corrected due to the size differences in mRNA inorder to make the doses equimolar.

TABLE 22 LNP Formulation Characterization DLin-KC2-DMA DSPC CholesterolPEG-DMG Mole Percent 50.0 10.0 38.5 1.5 (mol %)

TABLE 23 LNP Formulation Characterization Zeta Lipid: Mean atEncapsulation Drug mRNA size pH 7.4 (%) Concentration mRNA Ratio (nm)(mV) (Ribogreen) (mg/mL) Wild type 20:1 86.3 2.6 99 0.15 hGLP1 PDI: 0.081 × CTP 20:1 86.1 1.3 89 0.19 modified PDI: 0.04 hGLP1

TABLE 24 Dosing Regimen Injection Cationic Dose Size Ratios volume Lipid(mg/kg) to wt type (mL) Wild type hGLP1 KC2 0.05 1 0.2 1 × CTP modifiedhGLP1 KC2 0.07 1.4 0.2 PBS None None — 0.2

The rats were bled at 2 hours, 8 hours and terminally at 24 hours afteradministration to determine the human GLP1 protein expression in serumby ELISA. The serum levels of human GLP-1 protein expression in rat seraat different time points were measured by specific human GLP-1 ELISA andthe final conversion of pM to pg/ml calculated according to theconversion factor of the MW of engineered protein (conversion factor of3.3 for wild type GLP1, 8.85 for 1×CTP modified hGLP1 and 1 for PBS).

As shown in Table 25, human GLP1 protein expression was detectable inserum at least out to 24 hours with the group treated with 1×CTPmodified hGLP1 mRNA having the greatest performance.

TABLE 25 Serum Protein Expression in Rats Wild 1 × CTP 1 × CTP type Wildtype modified modified PBS hGLP1 hGLP1 hGLP1 hGLP1 (pg/ml) and (pg/ml)(pM) (pg/ml) (pM) (pM) 1 hour 98.9 30 763 86.2 22.7 6 hours 165.7 50.21234.8 139.5 32.8 12 hours 188.0 57 382.9 43.3 31.7

Example 20. Human GLP-1 Albumin and IgG4 Study

Albumin and IgG4 were evaluated as a potential approach for prolongingthe half-life of therapeutic protein molecules. mRNAs encoding humanGLP-1 were prepared using the in vitro transcription method for thesestudies.

Rats (n=6; Sprague Dawley rats with fitted jugular vein catheter(JVC-Harlan); approximate weight: 300-330 gm/rat) were administeredintravenously mRNA encoding the wild type sequence of human GLP1 (mRNAsequence shown in SEQ ID NO: 45; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (wild type hGLP1) formulatedin an LNP described in Table 26 (characterization in Table 27), mRNAencoding the human GLP1 sequence with albumin at C terminus (mRNAsequence shown in SEQ ID NO: 48; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (albumin modified hGLP1)formulated in an LNP described in Table 26 (characterization in Table27), mRNA encoding the human GLP1 sequence with IgG4 at C terminus (mRNAsequence shown in SEQ ID NO: 49; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) (IgG4 modified hGLP1)formulated in an LNP described in Table 26 (characterization in Table27) or a negative control of PBS (vehicle only) as outlined in Table 28.The dose of albumin modified hGLP1 and IgG4 modified hGLP1 was correcteddue to the size differences in mRNA in order to make the dosesequimolar.

TABLE 26 LNP Formulation Characterization DLin-KC2-DMA DSPC CholesterolPEG-DMG Mole Percent 50.0 10.0 38.5 1.5 (mol %)

TABLE 27 LNP Formulation Characterization Zeta Lipid: Mean atEncapsulation Drug mRNA size pH 7.4 (%) Concentration mRNA Ratio (nm)(mV) (Ribogreen) (mg/mL) Wild type 20:1 86.3 2.6 99 0.15 hGLP1 PDI: 0.08Albumin 20:1 77.2 1.2 90 0.12 modified PDI: 0.05 hGLP1 IgG4 20:1 87.02.4 98 0.14 modified PDI: 0.08 hGLP1

TABLE 28 Dosing Regimen Size Injection Cationic Ratio to Dose volumeLipid Cohort wt type (mg/kg) (mL) Wild type hGLP1 KC2 1 1 0.05 0.2Albumin modified hGLP1 KC2 2 4.98 0.25 0.2 IgG4 modified hGLP1 KC2 2 2.60.13 0.2 PBS none 1 — — 0.2

The rats administered wild type hGLP1 or PBS were bled at 2 hours, 8hours and terminally at 24 hours after administration, the ratsadministered albumin modified hGLP1 and IgG4 modified hGLP1 were bled at2 hours, 8 hours, 24 hours, 48 hours and 72 hours to determine the humanGLP1 protein expression in serum by GLP1 ELISA. The serum levels ofhuman GLP-1 protein expression in rat sera at different time points weremeasured by specific human GLP-1 ELISA and the final conversion of pM topg/ml calculated according to the conversion factor of the MW ofengineered protein (conversion factor of 3.3 for wild type GLP1, 36.5for albumin modified hGLP1, 30 for IgG4 modified hGLP1 and 1 for PBS).

As shown in Table 29, wild type hGLP1, albumin modified hGLP1 and IgG4modified hGLP1 expressed good amount of protein with the peak at 8hours. Albumin modified hGLP1 mRNA expressed very high protein levelthrough the time course curve until 72 hours mRNA and IgG4 modifiedhGLP1-IgG demonstrated a great superiority with half-life more than 72hours in rats. In Table 29, “NT” means not tested.

TABLE 29 Serum Protein Expression in Rats Albumin Albumin IgG4 IgG4 PBSWild type Wild type modified modified modified modified (pg/ml) hGLP1hGLP1 hGLP1 hGLP1 hGLP1 hGLP1 and (pg/ml) (pM) (pg/ml) (pM) (pg/ml) (pM)(pM) 2 hours 99 30 5938.6 162.7 4635 154.5 22.7 8 hours 165.7 50.241095.4 1125.9 41232 1374.4 32.8 24 hours 188.1 57 27287.4 747.6 28497949.9 31.7 48 hours NT NT 20889 572.3 29892 996.4 NT 72 hours NT NT5055.3 138.5 19080 636 NT

OTHER EMBODIMENTS

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

We claim:
 1. A method of expressing a fusion polypeptide in a humansubject, comprising administering to the human subject a lipidnanoparticle (LNP) comprising a codon optimized mRNA, wherein the mRNAencodes the fusion polypeptide, wherein the fusion polypeptide comprisesa secreted polypeptide operably linked at the carboxy terminus to analbumin or a functional fragment thereof, and wherein the mRNA is fullymodified at each uridine with 1-methylpseudouridine, and optionallymodified at each cytidine with 5-methylcytosine, thereby expressing thefusion polypeptide in the human subject.
 2. The method of claim 1,wherein the mRNA comprises a poly-A tail and wherein the poly-A tail isat least 140 nucleotides in length.
 3. The method of claim 1, whereinthe mRNA comprises a 5′ cap structure and wherein the 5′ cap structureis selected from the group consisting of: Cap0, Cap1, anti-reverse capanalog (ARCA), inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleicacid (LNA)-guanosine, and 2-azido-guanosine.
 4. The method of claim 1,wherein the fusion polypeptide comprises a signal peptide sequence. 5.The method of claim 1, wherein the albumin is human serum albumin.
 6. AnLNP comprising a codon optimized mRNA encoding a fusion polypeptide,wherein the fusion polypeptide comprises a secreted polypeptide operablylinked at the carboxy terminus to an albumin or a functional fragmentthereof, and wherein the mRNA is fully modified at each uridine with1-methylpseudouridine, and optionally modified at each cytidine with5-methylcytosine.
 7. A pharmaceutical composition comprising the LNP ofclaim 6 and a pharmaceutically acceptable excipient.
 8. The LNP of claim6, wherein the fusion polypeptide comprises a signal peptide sequence.9. The LNP of claim 6, wherein the albumin is human serum albumin.
 10. Amethod of increasing a level of a fusion polypeptide in a mammal,comprising administering to the mammal an LNP comprising a codonoptimized mRNA, wherein the mRNA encodes the fusion polypeptide, whereinthe fusion polypeptide comprises a secreted polypeptide operably linkedat the carboxy terminus to an albumin or a functional fragment thereof,and wherein the mRNA is fully modified at each uridine with1-methylpseudouridine, and optionally modified at each cytidine with5-methylcytosine, thereby increasing the level of the fusion polypeptidein the mammal.
 11. The method of claim 10, wherein the level of thefusion polypeptide is increased relative to the secreted polypeptide notfused at the carboxy terminus to the albumin or a functional fragmentthereof.
 12. The method of claim 10, wherein the level of the fusionpolypeptide is increased in serum or plasma of the mammal.
 13. Themethod of claim 12, wherein the level of the fusion polypeptide isdetermined by measuring the area under the curve (AUC) or the maximumserum or plasma concentration (C_(max)) of the fusion polypeptidefollowing administration to the mammal.
 14. The method of claim 10,wherein the fusion polypeptide comprises a signal peptide sequence. 15.The method of claim 10, wherein the albumin is human serum albumin.